Markers for cancer detection

ABSTRACT

The present invention relates to methods for detecting, prognosing and staging cancers, in particular cancers of the gastrointestinal tract. The methods of the invention comprise detecting specific protein markers in a tissue of interest, wherein the detected levels thereof may be indicative of pre-cancerous or cancerous tissue, or the stage or prognosis of a cancer. Further provided are methods of treating cancer, and cancer detection kits.

FIELD OF THE INVENTION

The present invention relates to methods for detecting, prognosing and staging cancers, in particular cancers of the gastrointestinal tract. The methods of the invention comprise detecting specific protein markers in a tissue of interest, wherein the detected levels thereof may be indicative of pre-cancerous or cancerous tissue, or the stage or prognosis of a cancer. Further provided are methods of using the markers for treating cancer, and cancer detection kits.

BACKGROUND OF THE INVENTION

Colorectal cancer, also referred to as colon cancer or large bowel cancer, is a malignant neoplastic disease associated with tumors in the colon, rectum and appendix. With 655,000 deaths worldwide per year, it is the third most common form of cancer and the second leading cause of cancer-related death in the Western world.

Colorectal cancers originate in the colorectal epithelium and typically are not extensively vascularized (and therefore not invasive) during the early stages of development. The transition to a highly vascularized, invasive and ultimately metastatic cancer, which spreads throughout the body, commonly takes ten years or longer. If the cancer is detected prior to invasion, surgical removal of the cancerous tissue is an effective cure. However, colorectal cancer is often detected only upon manifestation of clinical symptoms, such as pain and black tarry stool. Generally, such symptoms are present only when the disease is well established, often after metastasis has occurred, and the prognosis for the patient is poor, even after surgical resection of the cancerous tissue. For example, patients diagnosed with early colon cancer generally have a much greater, five-year survival rate as compared to the survival rate for patients diagnosed with distant metastasized colon cancer. Accordingly, early detection of colorectal cancer is of critical importance for reducing its morbidity.

Diagnostic methods for colon cancer most frequently depend on direct visual inspection of the gastrointestinal (GI) tract. Endoscopy involves inspection with a miniaturized light source at a probe end of a coherent bundle fiber optic cable. Reflected light beam images are returned through the fiber optic cable for detection by an external digital camera and display on an external monitor or for recording on an external video recorder or both. While this technique allows for identification, removal, and biopsy of potentially cancerous growths such as polyps, its use is associated with certain disadvantages, such as being expensive, uncomfortable, inherently risky due to its invasive nature, and the inability to access some portions of the large intestine and most of the small intestine.

Swallowable endoscopy capsules containing miniaturized optical, digital camera and radio transmission systems have been subsequently developed along with complementary external monitoring systems for inspecting the GI tract. For example, the capsule marketed under the trade name PillCam® SB was initially approved by the U.S. Food and Drug Administration in 2001 for detection and diagnosis of disorders of the small intestine. U.S. Pat. No. 5,604,531 discloses an in vivo video camera system comprising a swallowable capsule. The transit of endoscopy capsules through the small intestine is dependent on peristalsis, meaning that some areas with lesions may be missed if the capsule is not retained in that area for a sufficient amount of time. Further, the endoscopy capsules in current use are not capable of identifying molecular markers, which may be early indicators of colorectal cancer, even prior to the development of pre-cancerous polyps.

U.S. Pat. No. 7,468,044 discloses a system and method for in vivo and in situ detection of body lumen conditions, such as in the GI tract. The system comprises an interaction chamber comprising an indicator; a light source for illuminating the interaction chamber; and an optical detector for detecting in vivo optical changes occurring in the interaction chamber upon reaction of the indicator with an endo-luminal sample.

U.S. Pat. No. 7,515,953 discloses a method for detecting fluorescence emitted by cells in a wall of a body lumen, such as an intestinal wall, the method comprising use of a swallowable capsule, the capsule comprising a light source and a fluorescent-labeled probe, which is released from a reservoir in the capsule. According to the disclosure, an electric field generated from an electrode in the capsule enhances uptake of the probe, and a detector in the capsule detects the fluorescent signal emitted. By determining the intensity and/or position in the lumen wall of the fluorescent signal, a drug for killing abnormal cells is released from a second reservoir in the capsule. According to the disclosure, the abnormal cells may be cancer cells, colon polyps or precancerous cells.

U.S. Patent Application Publication No. 2008/0146896 discloses a device, such as an autonomous capsule, for in vivo analysis which includes a reaction chamber to store a detecting reagent able to react with a sample collected in vivo; and optionally a labeled-substance chamber to store a labeled substance able to bind to a compound resulting from a reaction of the detecting reagent and the sample. According to the disclosure, the detecting reagent may be an antibody.

U.S. Patent Application Publication No. 2009/0216082 discloses a device system for in vivo detection of target molecules in an endo-luminal sample, and a method for in vivo magnetic immunoassay, which may be used for the detection of cancer in the gastrointestinal tract.

Yet other methods of colon cancer detection are based on detection of particular proteins or genes which are considered to be specifically or differentially expressed in colon cancer.

U.S. Pat. No. 7,507,541 discloses a method of detecting the presence of inter alia colon cancer that is based on determining the level of 36P6D5 protein expressed by cells in a test tissue sample from an individual, and comparing the level to that expressed in a corresponding normal tissue sample.

U.S. Pat. No. 7,501,242 discloses a method of detecting colon cancer that is based on detecting levels of expression of tyrosine threonine kinase (TTK) in a test sample, such as a colon sample, that are increased by at least two fold relative to the level of expression in a normal non-cancer sample of the same type.

U.S. Pat. No. 7,452,727 discloses a automatable method for identifying cancer cells and their precursor cells that is based on detecting at least two molecular markers, wherein the detection of each marker alone is not a reliable indicator of the presence of cancer cells and their precursor cells. According to the disclosure, the molecular markers may be selected from her2/neu, Ki67, p53, her2/neu, bcl-2, MN, mdm-2, EGF receptor, bcl-2 and p16.

U.S. Pat. No. 7,402,403 discloses a method for the detection of cancer or early neoplastic change that is based on detecting autoantibodies directed to tumor marker antigens in a sample of bodily fluids, wherein the tumor marker antigens are selected from MUC1, p53, c-erbB2, Ras, c-myc, BRCA1, BRCA2, PSA, APC and CA125.

U.S. Pat. No. 7,129,043 discloses a method of identifying a human subject having an increased risk of developing colon cancer that is based on detecting upregulation of the CLN3 gene.

U.S. Pat. No. 7,115,368 relates to a method of detecting epithelial cancer cells inter alia colon cancer that is based on detection of expression in a biological sample of pellino proteins.

U.S. Pat. No. 7,098,008 relates to a method for detection of cancer inter alia colon cancer that is based on detecting expression of melanoma antigen gene (MAGE).

U.S. Pat. No. 7,078,180 relates to a method of diagnosing a cancer inter alia colon cancer that is based on detection of a ZEB (zfh-1/delta EF1) polypeptide.

U.S. Pat. No. 6,949,339 relates to methods for detecting, diagnosing, monitoring, staging, and prognosticating colon cancers, based on detection of Colon Specific Genes.

U.S. Pat. No. 6,919,176 discloses a method of detecting cancer inter alia colon cancer that is based on detection of expression of specific G-protein coupled receptors.

There remains an unmet need for methods of early detection, prognosis and treatment of colon cancer.

SUMMARY OF THE INVENTION

The present invention provides methods of detecting cancer that are based on the qualitative or quantitative identification of particular proteins, also referred to herein as molecular markers. Further provided are methods of cancer prevention, prognosis and treatment.

Disclosed herein for the first time is a specific group of protein markers which are indicative of both pre-cancerous and cancerous lesions. Further disclosed herein for the first time is an additional specific group of protein markers which are primarily indicative of pre-cancerous lesions.

The invention is based in part, on the unexpected discovery that the level of expression within the gastrointestinal tract of certain proteins is significantly increased in both pre-cancerous and cancerous tissue relative to the level of expression of the same proteins in healthy tissue of the same type Surprisingly, the expression of these markers is elevated, even compared to healthy tissue bordering tumor growth. It has also been surprisingly found that the expression of yet other markers is significantly increased in early stage cancer, and significantly decreased in later stages of cancer

Without wishing to be bound by any particular theory or mechanism of action, the invention enables identification of individuals at risk of developing cancer, in particular colorectal cancer, even prior to observable histological changes in affected tissue. Since the methods of the invention are based on changes in protein expression patterns in cells, rather than later occurring pathological changes in tissue, a level of sensitivity is obtained that is greater by many orders of magnitude than current techniques of cancer detection. Thus, in the case of colorectal cancer, the invention provides a means of predicting the disease well in advance of the possibility of detecting potentially cancerous polyps by conventional endoscopic examination, the latter being the current yet inadequate standard for early detection.

The principles of the current invention are exemplified herein by quantitative mass spectroscopy analysis of healthy, pre-cancerous and cancerous tissue obtained from the gastrointestinal tract of patients during surgical excision of early stage (e.g. polyps) or more advanced stage tumors, which has resulted in the identification of a specific group of proteins, the expression of each of which is significantly increased in diseased colon tissue, as compared to healthy colon tissue. This group of proteins, which includes KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20), has not been previously disclosed, either in part or as a whole, to be useful for detection of colorectal cancer at any stage of the disease.

It is to be specifically understood however, that the current method of the invention need not be limited to examination of colon tissue obtained by surgical means, nor should it be limited to detection and quantification of the subject molecular markers using mass spectrometry techniques. Rather, the invention may be advantageously practiced using for example immunological techniques and reagents for detection of the subject molecular markers, either in vivo or ex vivo. For example, labeled monoclonal antibodies can be used for in vivo detection and quantitation of such proteins in different tissue compartments and regions. The detection may be accomplished for example, using pharmaceutical compositions or endoscopy probes which incorporate specifically designed chemical, immunological or nucleic acid reagents. Advantageously, labeled reagents such as antibodies, which specifically interact with the subject molecular markers may be prepared as injectable or ingestible pharmaceutical compositions and following administration the interaction with their molecular targets may be externally monitored. Alternately or in addition, the invention may be practiced by analysis of biological samples obtained from a subject, such as biopsy tissue.

In a first aspect, the invention provides a method of detecting cancer in a subject, the method comprising: (i) detecting in a biological sample from the subject at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20), so as to determine the level of the at least one protein; and (ii) comparing the level determined in (i) to a reference level of the same at least one protein, wherein detection of a level of said at least one protein in the biological sample which is significantly different from the reference level, is indicative of cancer in the subject.

Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.

In another aspect, the invention provides a method for determining the stage of a cancerous or pre-cancerous growth in a subject, the method comprising: (i) detecting in a test sample from the subject at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20), so as to determine the level of the at least one protein; and (ii) comparing the level determined in (i) to a reference level of the same at least one protein; wherein the level detected in the test sample is indicative of the stage of the growth.

Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.

In another aspect, the invention provides a method for determining the prognosis of a cancerous disease in a subject, the method comprising: (i) detecting in a test sample from the subject at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20), so as to determine the level of the at least one protein; and (ii) comparing the level determined in (i) to a reference level of the same at least one protein; wherein the level detected in the test sample is indicative of the prognosis of the cancerous disease.

Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.

In particular embodiments, the biological sample is selected from the group consisting of blood, serum, nipple aspirate fluid, lymph node aspirate, a biopsy sample, a tumor sample, a tissue sample, mucosal fluid, cervical wash, lacrimal duct fluid, urine, saliva, pleural effusion and sputum. Each of the aforementioned materials represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.

In currently preferred embodiments, the biological sample comprises a tissue sample. In currently preferred embodiments, the tissue sample comprises gastrointestinal tissue, particularly colorectal tissue or pre-cancerous tissue such as polyps. In currently preferred embodiments, the biological sample is selected from a tumor within the gastrointestinal tract, particularly a colorectal tumor. In particular embodiments, the gastrointestinal tissue is from an area or organ selected from the group consisting of the esophagus, the stomach, the small intestine, the large intestine (colon), the rectum, the appendix and a combination thereof.

In particular embodiments, the cancer being detected is selected from the group consisting of adrenal cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, fallopian tube cancer, gastric cancer, head and neck cancer, hepatic cancer, lung cancer including small cell lung cancer and non-small cell lung cancer, melanoma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, thyroid and parathyroid cancer, renal cancer, sarcoma, thymoma, hematological malignancies and germ cell tumors. In a currently preferred embodiment, the cancer is colorectal cancer. Each of the aforementioned cancers represents a separate embodiment of the invention and may be used independently from any of the others.

In a particular embodiment, the at least one protein detected is indicative of a disorder selected from the group consisting of pre-cancerous polyps, early stage colorectal cancer and advanced stage colorectal cancer. In particular embodiments, a method of detecting pre-cancerous polyps or early stage colorectal cancer comprises detecting at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20); and further comprises detecting at least one protein selected from the group consisting of CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49) and CISD1 (SEQ ID NO:50). Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.

In particular embodiments, a method of detecting pre-cancerous polyps or early stage colorectal cancer comprises detecting at least one of KIAA0152 (SEQ ID NO:1) and NAMPT (SEQ ID NO:2); and further comprises detecting at least one protein selected from the group consisting of CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49) and CISD1 (SEQ ID NO:50). Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.

In particular embodiments, a method of the invention comprises use of at least one reagent suitable for detecting the level of at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20).

In particular embodiments, said reagent is suitable for detecting the level of at least one of KIAA0152 (SEQ ID NO:1) and NAMPT (SEQ ID NO:2). Each of the aforementioned reagents represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.

In particular embodiments, a method of the invention further comprises use of at least one reagent suitable for detecting the level of at least one protein selected from the group consisting of CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49) and CISD1 (SEQ ID NO:50). Each of the aforementioned reagents represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.

In particular embodiments, the biological sample or test sample is obtained from the subject by a procedure selected from the group consisting of biopsy, flexible endoscopy, double balloon endoscopy and surgical colorectal re-sectioning.

In particular embodiments, the biological sample or test sample is assessed in vivo in the subject. In particular embodiments, the method comprises contacting a body tissue, cavity or fluid with at least one of a pharmaceutical composition and an endoscopy apparatus.

In particular embodiments, the method comprises contacting a body tissue, cavity or fluid with at least one reagent suitable for detecting the level of at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20)₄. In particular embodiments, the body tissue, cavity or fluid is contacted with a reagent is suitable for detecting the level of at least one of KIAA0152 (SEQ ID NO:1) and NAMPT (SEQ ID NO:2). Each of the aforementioned reagents represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.

In particular embodiments, the method further comprises contacting a body tissue, cavity or fluid with at least one reagent suitable for detecting the level of at least one protein selected from the group consisting of CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49) and CISD1 (SEQ ID NO:50). Each of the aforementioned reagents represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.

In particular embodiments, the method comprises administering a diagnostic pharmaceutical composition. In particular embodiments, the administering of the pharmaceutical composition is by a route selected from the group consisting of oral, parenteral, subcutaneous, intramuscular, intrathoracic and intraarticular.

In particular embodiments, the pharmaceutical composition or the endoscopy apparatus comprise at least one reagent suitable for detecting the level of at least one of the aforementioned proteins. In particular embodiments, the pharmaceutical composition or the endoscopy apparatus comprise at least one reagent suitable for detecting the level of at least one of KIAA0152 (SEQ ID NO:1) and NAMPT (SEQ ID NO:2).

In particular embodiments, the reagent specifically interacts with at least one of the aforementioned proteins or with nucleic acid encoding at least one of the aforementioned proteins, or a fragment of said protein. In particular embodiments, the pharmaceutical composition or the endoscopy apparatus comprises a multiplicity of reagents, wherein each reagent of the multiplicity specifically interacts with one distinct protein or with nucleic acid encoding one distinct protein or a fragment thereof. In particular embodiments, the reagent is selected from an antibody, an antibody mimetic and a nucleic acid. In particular embodiments, the antibody is selected from a monoclonal antibody, a humanized antibody, a single chain antibody, an antibody fragment and combinations thereof. In particular embodiments, the pharmaceutical composition or the endoscopy apparatus comprise a multiplicity of antibody mimetics, wherein each antibody mimetic of the multiplicity specifically interacts with one of the aforementioned proteins.

In particular embodiments, the reagent comprises a detectable label, such as a fluorescent label, a radiolabel or an enzymatic label.

In particular embodiments, the pharmaceutical composition or the endoscopy apparatus comprise at least one least one nucleic acid, wherein the at least one nucleic acid is complementary to a nucleic acid encoding at least one of the aforementioned proteins, or a fragment of said protein. In particular embodiments, the at least one nucleic acid comprises a multiplicity of nucleic acids, wherein each nucleic acid of the multiplicity is complementary to a nucleic acid encoding one distinct protein of the aforementioned proteins, or a fragment of said protein. In particular embodiments, the nucleic acid comprises a detectable label, such as a fluorescent label, a radiolabel or an enzymatic label.

In particular embodiments, the detecting in step (i) comprises use of an assay system. In particular embodiments, the assay system comprises an immunoassay, a nucleic acid hybridization assay, a binding assay, an array, a phage display library or combinations thereof. In particular embodiments, the array is a protein array, or a phage display library array. In particular embodiments, the assay system comprises at least one reagent suitable for detecting the level of at least one of the aforementioned proteins, as described herein. In particular embodiments, the assay system comprises a multiplicity of such reagents, for example antibodies, wherein each reagent in the multiplicity specifically interacts with one of the aforementioned proteins.

In particular embodiments, the detecting comprises use of an external monitoring system. In particular embodiments, the external monitoring system is configured to display the level of the at least one protein detected.

In particular embodiments, a method of the invention further comprises detecting in the sample at least one protein selected from the group consisting of FAM62B (SEQ ID NO:51), SLC1A5 (SEQ ID NO:52), RSL1D1 (SEQ ID NO:53), LYZ (SEQ ID NO:54), THBS1 (SEQ ID NO:55), LMO7 (SEQ ID NO:56), TNC (SEQ ID NO:57), RBM39 (SEQ ID NO:58), ILVBL (SEQ ID NO:59), ERO1L (SEQ ID NO:60), LOC442497 (SEQ ID NO:61), TCOF1 (SEQ ID NO:62), SERPINB9 (SEQ ID NO:63), HSDL2 (SEQ ID NO:64), ADAMDEC1 (SEQ ID NO:65), AMACR (SEQ ID NO:66), AMACR;C1QTNF3 (SEQ ID NO:67), ARID1A (SEQ ID NO:68), CEBPZ (SEQ ID NO:69), COL5A1 (SEQ ID NO:70), EFEMP2 (SEQ ID NO:71), FAM84B (SEQ ID NO:72), FKBP10 (SEQ ID NO:73), FKBP9 (SEQ ID NO:74), GPRC5A (SEQ ID NO:75), KPNA2 (SEQ ID NO:76), MMP1 (SEQ ID NO:77), PNMA5 (SEQ ID NO:78), POLR1C (SEQ ID NO:79), SPARC (SEQ ID NO:80), UBAP2 (SEQ ID NO:81), UCK2 (SEQ ID NO:82) and WDR74 (SEQ ID NO:83). Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.

In particular embodiments, a method of the invention further comprises use of at least one reagent suitable for detecting the level of at least one protein selected from the group consisting of FAM62B (SEQ ID NO:51), SLC1A5 (SEQ ID NO:52), RSL1D1 (SEQ ID NO:53), LYZ (SEQ ID NO:54), THBS1 (SEQ ID NO:55), LMO7 (SEQ ID NO:56), TNC (SEQ ID NO:57), RBM39 (SEQ ID NO:58), ILVBL (SEQ ID NO:59), ERO1L (SEQ ID NO:60), LOC442497 (SEQ ID NO:61), TCOF1 (SEQ ID NO:62), SERPINB9 (SEQ ID NO:63), HSDL2 (SEQ ID NO:64), ADAMDEC1 (SEQ ID NO:65), AMACR (SEQ ID NO:66), AMACR;C1QTNF3 (SEQ ID NO:67), ARID (SEQ ID NO:68), CEBPZ (SEQ ID NO:69), COL5A1 (SEQ ID NO:70), EFEMP2 (SEQ ID NO:71), FAM84B (SEQ ID NO:72), FKBP10 (SEQ ID NO:73), FKBP9 (SEQ ID NO:74), GPRC5A (SEQ ID NO:75), KPNA2 (SEQ ID NO:76), MMP1 (SEQ ID NO:77), PNMA5 (SEQ ID NO:78), POLR1C (SEQ ID NO:79), SPARC (SEQ ID NO:80), UBAP2 (SEQ ID NO:81), UCK2 (SEQ ID NO:82) and WDR74 (SEQ ID NO:83). Each of the aforementioned reagents represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.

In particular embodiments, the cancer is a cancer other than colorectal cancer and the at least one protein includes at least one of FAM62B (SEQ ID NO:51), SLC1A5 (SEQ ID NO:52), RSL1D1 (SEQ ID NO:53), LYZ (SEQ ID NO:54), TNC(SEQ ID NO:57), RBM39 (SEQ ID NO:58), ERO1L (SEQ ID NO:60), LOC442497 (SEQ ID NO:61), TCOF1 (SEQ ID NO:62), NAMPT (SEQ ID NO:2), SERPINB9 (SEQ ID NO:63), HSDL2 (SEQ ID NO:64).

In particular embodiments, the level of the at least one protein in the biological or test sample is increased by at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 20-fold, or at least 50-fold, relative to the reference level. In particular embodiments, the reference level is representative of the level of the same protein in non-diseased tissue. In particular embodiments, the reference level is representative of the level of the same protein in a particular stage or form of cancer. In particular embodiments, the reference level is representative of the level of the same protein in a cancer having a known prognosis.

In another aspect, the invention provides a method of treating cancer, the method comprising administering to a subject in need thereof at least one pharmaceutical agent, wherein the pharmaceutical agent specifically interacts with at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19), CCT4 (SEQ ID NO:20), CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49), CISD1 (SEQ ID NO:50), LYZ (SEQ ID NO:54), LOC442497;SLC3A2 (SEQ ID NO:61), DMBT1 (SEQ ID NO:84), NUCB1 (SEQ ID NO:85), GGH (SEQ ID NO:86), AGR3 (SEQ ID NO:87), TM9SF2 (SEQ ID NO:88), SYK (SEQ ID NO:89), GCA (SEQ ID NO:90), HDLBP (SEQ ID NO:91), C1QBP (SEQ ID NO:92) and CLIC1 (SEQ ID NO:93). Each of the aforementioned agents represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.

In a particular embodiment, the at least one protein is selected from the group consisting of KIAA0152 (SEQ ID NO:1), LYZ (SEQ ID NO:54), LOC442497;SLC3A2 (SEQ ID NO:61), DMBT1 (SEQ ID NO:84), NUCB1 (SEQ ID NO:85), GGH (SEQ ID NO:86), AGR3 (SEQ ID NO:87), TM9SF2 (SEQ ID NO:88), SYK (SEQ ID NO:89), GCA (SEQ ID NO:90), HDLBP (SEQ ID NO:91), C1QBP (SEQ ID NO:92) and CLIC1 (SEQ ID NO:93). In particular embodiments, the pharmaceutical agent specifically interacts with KIAA0152 (SEQ ID NO:1). Each of the aforementioned reagents represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.

In particular embodiments, the pharmaceutical agent is selected from an antibody and an antibody mimetic. In particular embodiments, the pharmaceutical agent further comprises a cytotoxic moiety, such as a plant toxin, a bacterial toxin, a radioactive moiety or a chemotherapeutic agent. In particular embodiments, the pharmaceutical agent is selected from a chemical conjugate and fusion protein.

In another aspect, the invention provides an antigen composition comprising at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19), CCT4 (SEQ ID NO:20), CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49), CISD1 (SEQ ID NO:50), LYZ (SEQ ID NO:54), LOC442497;SLC3A2 (SEQ ID NO:61), DMBT1 (SEQ ID NO:84), NUCB1 (SEQ ID NO:85), GGH (SEQ ID NO:86), AGR3 (SEQ ID NO:87), TM9SF2 (SEQ ID NO:88), SYK (SEQ ID NO:89), GCA (SEQ ID NO:90), HDLBP (SEQ ID NO:91), C1QBP (SEQ ID NO:92) and CLIC1 (SEQ ID NO:93), or an immunogenic fragment of said at least one protein. Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.

In a particular embodiment, the composition comprises at least one of CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49) and CISD1 (SEQ ID NO:50), or an immunogenic fragment thereof. Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.

In a particular embodiment, the composition comprises KIAA0152 (SEQ ID NO:1) or an immunogenic fragment thereof. In a particular embodiment, a method of preventing or treating cancer in a subject in need thereof comprises administering to the subject an effective amount of said antigen composition. Further provided is an antigen composition as previously specified; for use in preventing or treating cancer.

In another aspect, the invention provides a kit for detecting, staging or prognosing cancer, the kit comprising at least one reagent suitable for detecting the level of at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19), CCT4 (SEQ ID NO:20); CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49) and CISD1 (SEQ ID NO:50). Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.

In particular embodiments, the kit is for detecting pre-cancerous polyps or early stage colorectal cancer and comprises at least one reagent suitable for detecting the level of at least one protein selected from the group consisting of CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49) and CISD1 (SEQ ID NO:50). In particular embodiments, the kit comprises a reagent suitable for detecting the level of KIAA0152 (SEQ ID NO:1).

According to some embodiments the present invention excludes proteins known to be associated with colorectal cancers. According to other embodiments the methods of the present invention exclude proteins that were known to be associated with other types of cancers.

Other objects, features and advantages of the present invention will become clear from the following description and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates methods used for the identification of the protein diagnostic markers of cancer according to the invention.

FIG. 2 illustrates quantitative data analysis of protein markers identified in diseased and non-diseased tissues from human subjects with colorectal cancer, including patients with polyps. Sample numbers indicated along the top of the figure correspond to different patients, and in some cases different samples from a single patient. Proteins are indicated along the left side. The height of each rectangle corresponds to the relative signal intensity, and the shading corresponds to the quantitative ratio (protein in diseased tissue:protein in non-diseased tissue) from the same patient. Black shaded rectangles, ratio>10; white rectangles, ratio in the range 3-10; gray shaded rectangles, ratio in the range 0.3-3; diamond filled rectangles, ratio<0.3.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of identification of cancerous cells by detection of levels of particular proteins, also referred to herein as “cancer-associated proteins or “molecular markers”, which have been found to be differentially expressed in cancer tissue, in particular, colorectal cancer tissue. Details of the subject cancer-associated proteins disclosed herein are provided in Tables 1 and 3-7. Some of the identified proteins for which significantly elevated levels were detected in colorectal cancer tissue as compared to normal colorectal tissue have not been previously disclosed to be associated with cancerous disease. Examples of such proteins are indicated in Table 1 as denoted by the symbol “+” in the column labeled “Newly identified as cancer-associated” and include the proteins FAM62B, SLC1A5. RSL1D1, LYZ, TNC, RBM39, ILVBL, ERO1L, LOC442497, TCOF1, NAMPT, SERPINB9 and HSDL2. Yet other proteins, in particular THBS1 and LMO7, have been disclosed in the prior art to be down-regulated in cancerous conditions, whereas the inventors of the present invention now disclose significantly increased levels of these proteins in cancer tissue, indicative of up-regulation. These proteins are indicated in Table 1 as denoted by the symbol “UR” in the column labeled “Newly identified as cancer-associated”. Yet other proteins were detected at significant levels in colorectal cancer tissue but were completely absent from healthy colorectal tissue in the same patients. Such proteins are indicated in Table 1 as denoted by the symbol “+” in the column labeled “Detected in cancerous but not healthy” and include the proteins AMACR, AMACR;C1 QTNF3, COL5A1, FKBP10, FKBP9, GPRC5A, POLR1c, SPARC and UBAP2.

Table 3 list proteins which are highly expressed in colorectal cancer, both in pre-cancerous polyps and in more advanced stages of the disease. Accordingly, any of the proteins listed in Table 3 represent candidates for use as diagnostic markers of colorectal cancer, which can be indicative of any of pre-cancerous lesions, early stage or late stage forms of the disease. Two currently preferred protein markers are KIAA0152 and NAMPT.

A quantitative analysis of some of the proteins listed in Table 3 is shown in FIG. 2. The analysis shows that in a large number of the patients examined, the quantitative ratio of protein in diseased tissue (i.e. polyps and/or cancer): protein in non-diseased tissue from the same patient, is 3 or greater.

Table 4 lists proteins which appear to be highly expressed in pre-cancerous polyps, yet their expression tends to be diminished in more advanced stages of the disease. Accordingly, proteins listed in Table 4 represent promising candidates for use as very early diagnostic markers to identify individuals at risk of developing colorectal cancer.

Identification and quantification of a panel of tumor-associated proteins such as those listed in Tables 1 and 3-6, provides a specific means of detecting colorectal cancer in a subject, as well as means of prognosing and staging previously diagnosed colorectal cancers. Such methods can conveniently be carried out using detectably-labeled reagents which specifically bind the selected target proteins. Typically such reagents comprise monoclonal antibodies or small chemical mimetics thereof. In some cases however, it may be advantageous to employ nucleic acids complementary to mRNA species encoding the target proteins so as to quantitate expression activity which may be correlated with the corresponding protein levels. Furthermore, therapeutic pharmaceutical agents directed against tumor-associated proteins, for example, those disclosed in Table 7 herein, may be prepared, for example monoclonal antibodies conjugated to cytotoxic moieties, for use in targeted therapy of colorectal cancer.

TABLE 1 Detected Newly in IPI identified cancerous Acc. as cancer- but not Protein Name No. associated healthy ABCF2 ATP-binding cassette, sub- 68506 family F, member 2 isoform b ACIN1 Isoform 1 of Apoptotic 7334 chromatin condensation inducer in the nucleus ACOT7 Isoform 1 of Cytosolic acyl 10415 coenzyme A thioester hydrolase ADAMDEC1 ADAM DEC1 4480 + precursor ADO 2-aminoethanethiol 45939 dioxygenase ADSS Adenylosuccinate synthetase 26833 isozyme 2 AK3 GTP:AMP phosphotransferase 465256 mitochondrial ALG5 Dolichyl-phosphate beta- 2506 glucosyltransferase AMACR Alpha-methylacyl-CoA 847727 + racemase AMACR; C1QTNF3 alpha- 5918 + methylacyl-CoA racemase isoform 1 ANXA3 Annexin A3 24095 ARHGEF1 Isoform 1 of Rho guanine 647786 nucleotide exchange factor 1 ARID1A Isoform 1 of AT-rich 643722 + interactive domain-containing protein 1A ARRDC1 Arrestin domain containing 1 514937 ATAD3A Isoform 2 of ATPase 295992 family AAA domain-containing protein 3A ATP6AP2 Renin receptor precursor 168884 ATP6V0A1 97 kDa protein 892784 ATP6V1E1 vacuolar H+ ATPase E1 719806 isoform b BMS1 Ribosome biogenesis protein 6099 BMS1 homolog BPI Bactericidal/permeability- 552280 increasing protein BUD31 Protein BUD31 homolog 13180 BXDC1 Brix domain containing 1 644504 C14orf21 Pumilio domain-containing 216999 protein C14orf21 C1orf116 Isoform 1 of Specifically 28392 androgen-regulated gene protein C1R; ACYP1; C17orf13 Complement 296165 C1r subcomponent precursor C20orf43 UPF0549 protein C20orf43 297121 C2orf47 Uncharacterized protein 291751 C2orf47, mitochondrial precursor C3orf64 Isoform 1 of 396231 Uncharacterized glycosyltransferase AER61 precursor C7orf24 Uncharacterized protein 31564 C7orf24 C8orf55 Uncharacterized protein 171421 C8orf55 precursor CAD Putative uncharacterized protein 893035 CAD CASP8 Uncharacterized protein 220725 CASP8 CBFB Core-binding factor subunit 16746 beta CEACAM5 Carcinoembryonic 27486 antigen-related cell adhesion molecule 5 precursor CEBPZ CCAAT/enhancer-binding 306723 + protein zeta CHD4 Isoform 2 of Chromodomain- 455210 helicase-DNA-binding protein 4 CLPB Isoform 2 of Caseinolytic 216192 peptidase B protein homolog COL12A1 Isoform 1 of Collagen 329573 alpha-1(XII) chain precursor COL5A1 Collagen alpha-1(V) chain 844090 + precursor COMT Isoform Soluble of Catechol 375513 O-methyltransferase CSTF1 Cleavage stimulation factor 11528 50 kDa subunit CTSG Cathepsin G precursor 28064 DCK Deoxycytidine kinase 20454 DDX18 ATP-dependent RNA 301323 helicase DDX18 DEK 48 kDa protein 871695 DHCR7 7-dehydrocholesterol 294501 reductase DHODH Dihydroorotate 24462 dehydrogenase, mitochondrial precursor DHX30 DEAH (Asp-Glu-Ala-His) 477295 box polypeptide 30 isoform 2 DIAPH1 Diaphanous homolog 1 884341 DMBT1 Isoform 1 of Deleted in 99110 malignant brain tumors 1 protein precursor DNAJA3 Isoform 2 of DnaJ homolog 179187 subfamily A member 3, mitochondrial precursor DNAJC19 10 kDa protein 795263 DPEP1 Dipeptidase 1 precursor 59476 EFEMP2 Mutant p53 binding protein 556657 + 1 variant (Fragment) EI24 Isoform 2 of Etoposide-induced 23185 protein 2.4 homolog EIF2B3 Isoform 1 of Translation 6504 initiation factor eIF-2B subunit gamma EIF2S2 Eukaryotic translation 21728 initiation factor 2 subunit 2 ERO1L ERO1-like protein alpha 386755 + precursor EXOC2 Exocyst complex component 2 783559 F11R Junctional adhesion molecule A 1754 precursor FAM62B Isoform 2 of Extended 409635 + synaptotagmin-2 FAM84B Protein FAM84B 64666 + FAP Isoform 1 of Seprase 295461 FASN Fatty acid synthase 26781 FASTKD5 FAST kinase domain- 414973 containing protein 5 FDFT1 Squalene synthetase 20944 FERMT1 Isoform 1 of Fermitin 304754 family homolog 1 FKBP10 FK506-binding protein 10 303300 + precursor FKBP9 FK506-binding protein 9 182126 + precursor FOXK1 Isoform 1 of Forkhead box 556645 protein K1 FRG1 Protein FRG1 4655 FYB FYN binding protein (FYB- 73110 120/130) isoform 1 GCA Grancalcin 4524 GEMIN5 Gem-associated protein 5 291783 GGH Gamma-glutamyl hydrolase 23728 precursor GLRX3 Glutaredoxin-3 8552 GLT25D1 Glycosyltransferase 25 168262 family member 1 precursor GMPS GMP synthase 29079 GNL3 Isoform 1 of Guanine 306380 nucleotide-binding protein-like 3 GPR89B; GPR89A Isoform 1 of 472858 Protein GPR89 GPRC5A Retinoic acid-induced 22624 + protein 3 GPX2 Glutathione peroxidase 2 298176 GTF2F1 General transcription factor 17450 IIF subunit 1 GTF2I Isoform 2 of General 293242 transcription factor II-I HCFC1 Uncharacterized protein 641743 HCFC1 HM13 Isoform 1 of Minor 152441 histocompatibility antigen H13 HSDL2 Isoform 1 of Hydroxysteroid 414384 + dehydrogenase-like protein 2 HSPH1 Isoform Beta of Heat shock 218993 Protein 105 kDa ICAM1 Intercellular adhesion 8494 molecule 1 precursor IGFBP7 Insulin-like growth factor- 16915 binding protein 7 precursor IKIP IKIP2 401791 ILVBL Isoform 1 of Acetolactate 554541 synthase-like protein IPO7 Importin-7 7402 ISLR Immunoglobulin superfamily 23648 containing leucine-rich repeat protein precursor KIAA0020 Pumilio domain- 791325 containing protein KIAA0020 KIAA0241 Isoform 1 of Protein 397348 KIAA0241 KIAA1219 Isoform 3 of Protein 410120 KIAA1219 KPNA2 Karyopherin alpha 2 789457 + LACTB2 Beta-lactamase-like protein 2 6952 LCN2 Lipocalin 2 643623 LEPRE1 Isoform 3 of Prolyl 3- 45839 hydroxylase 1 precursor LMO7 Isoform 3 of LIM domain only 291802 UR protein 7 LOC442497; SLC3A2 solute carrier 554722 + family 3 (activators of dibasic and neutral amino acid transport), member 2 isoform e LOC731605 similar to BCL2- 886854 associated transcription factor 1 isoform 2 LTBP2 Latent-transforming growth 292150 factor beta-binding protein 2 precursor LTF Lactotransferrin precursor 848342 LYZ Lysozyme C precursor 19038 + MAN1A2 Uncharacterized protein 743100 MAN1A2 MCM2 DNA replication licensing 184330 factor MCM2 MCM3 DNA replication licensing 13214 factor MCM3 MCM5 DNA replication licensing 18350 factor MCM5 MCM6 DNA replication licensing 31517 factor MCM6 MCM7 Isoform 1 of DNA replication 299904 licensing factor MCM7 MMP1 Interstitial collagenase 8561 + precursor MMP2 72 kDa type IV collagenase 27780 precursor MMP9 Matrix metalloproteinase-9 27509 precursor MOCS3 Molybdenum cofactor 4489 synthesis protein 3 MPO Isoform H7 of Myeloperoxidase 236556 precursor MRPS6 Mitochondrial 28S ribosomal 305668 protein S6 MTA2 Metastasis-associated protein 171798 MTA2 MUC16 Mucin-16 103552 NAMPT Isoform 1 of Nicotinamide 18873 + phosphoribosyltransferase NAT10 N-acetyltransferase 10 300127 NCBP1 Nuclear cap-binding protein 19380 subunit 1 NCF4 Isoform 1 of Neutrophil 14338 cytosol factor 4 NEBL Nebulette variant 4 872370 NIP7 Isoform 1 of 60S ribosome 7175 subunit biogenesis protein NIP7 homolog NOC2L Nucleolar complex protein 2 411886 homolog NOC4L Nucleolar complex protein 4 31661 homolog NOL10 Isoform 1 of Nucleolar 29513 protein 10 NQO1 NAD 12069 NUDCD1 Isoform 2 of NudC 306398 domain-containing protein 1 NUP188 Isoform 1 of Nucleoporin 477040 NUP188 homolog NUP210 Isoform 1 of Nuclear pore 291755 membrane glycoprotein 210 precursor NXF1 Nuclear RNA export factor 1 33153 OCIAD2 Isoform 1 of OCIA domain- 555902 containing protein 2 OLFM4 Olfactomedin-4 precursor 22255 OSBPL2 Isoform 1 of Oxysterol- 14137 binding protein-related protein 2 OTUD6B OTU domain containing 182180 6B PAICS Multifunctional protein ADE2 217223 PARP14 poly (ADP-ribose) 291215 polymerase family, member 14 PCNA Proliferating cell nuclear 21700 antigen PEX14 Peroxisomal membrane 25346 protein PEX14 PHF6 Isoform 1 of PHD finger 395568 protein 6 PKP3 Plakophilin-3 26952 PLOD3 Procollagen-lysine, 2- 30255 oxoglutarate 5-dioxygenase 3 precursor PNMA5 Paraneoplastic antigen-like 514588 + protein 5 POLR1C Isoform 1 of DNA-directed 5179 + RNA polymerases I and III subunit RPAC1 PROM1 Prominin-1 precursor 12540 PSAT1 Isoform 1 of Phosphoserine 1734 aminotransferase PTDSS1 Phosphatidylserine synthase 1 10746 PTK7 Tyrosine-protein kinase-like 7 298292 precursor PUF60 Isoform 5 of Poly 856076 PYCR1 pyrroline-5-carboxylate 376503 reductase 1 isoform 2 PYCR2 Pyrroline-5-carboxylate 470610 reductase 2 RAD21 Double-strand-break repair 6715 protein rad21 homolog RANBP3 Isoform 2 of Ran-binding 456728 protein 3 RBM12 RNA-binding protein 12 550308 RBM39 Isoform 2 of RNA-binding 215801 + protein 39 RCC1 regulator of chromosome 787306 condensation 1 isoform b RCC2 Protein RCC2 465044 RCN1 Reticulocalbin-1 precursor 15842 RDBP Negative elongation factor E 858 REEP6 Receptor expression- 647161 enhancing protein 6 REG1A Lithostathine-1-alpha 9027 precursor REG1B Lithostathine-1-beta 9197 precursor RFC2 Isoform 2 of Replication factor 218280 C subunit 2 RFC5 Replication factor C subunit 5 31514 RRM1 Ribonucleoside-diphosphate 13871 reductase large subunit RRP1 RRP1-like protein 550766 RRS1 Ribosome biogenesis 14253 regulatory protein homolog RSL1D1 RSL1D1 protein 642046 + S100A11 Protein S100-A11 13895 S100A12 Protein S100-A12 218131 S100A8 Protein S100-A8 7047 S100A9 Protein S100-A9 27462 SAE1 SUMO-activating enzyme 33130 subunit 1 SDCCAG10 Isoform 1 of Peptidyl- 25174 prolyl cis-trans isomerase SDCCAG10 SERPINB5 Serpin B5 precursor 783625 SERPINB9 Serpin B9 32139 + SERPINH1 Serpin H1 precursor 32140 SET Isoform 2 of Protein SET 301311 SLC1A5 Neutral amino acid 19472 + transporter B SLC25A15 Mitochondrial ornithine 3389 transporter 1 SLC2A1 Solute carrier family 2, 220194 facilitated glucose transporter member 1 SORD 11 kDa protein 791243 SPARC SPARC precursor 14572 + SQSTM1 Isoform 1 of 179473 Sequestosome-1 SRM Spermidine synthase 292020 SRRM2 Isoform 1 of Serine/arginine 782992 repetitive matrix protein 2 SSBP1 Single-stranded DNA-binding 29744 protein, mitochondrial precursor SYK Isoform Long of Tyrosine- 18597 protein kinase SYK TBC1D2B Isoform 1 of TBC1 550733 domain family member 2B TCOF1 Isoform 2 of Treacle protein 298696 + TFRC Transferrin receptor protein 1 22462 TH1L Isoform NELF-D of Negative 759539 elongation factor C/D THBS1 Thrombospondin-1 precursor 296099 UR THBS2 Thrombospondin-2 precursor 18769 THOC6 Isoform 1 of THO complex 328985 subunit 6 homolog TIMP1 TIMP metallopeptidase 642739 inhibitor 1 TJP2 Isoform A1 of Tight junction 3843 protein ZO-2 TM9SF4 Isoform 2 of 885106 Transmembrane 9 superfamily member 4 precursor TNC Isoform 1 of Tenascin precursor 31008 + TOMM34 Mitochondrial import 9946 receptor subunit TOM34 TOP1 DNA topoisomerase 1 413611 TOP2A Isoform 2 of DNA 414101 topoisomerase 2-alpha TPR nuclear pore complex-associated 742682 protein TPR TRMT6 Isoform 1 of tRNA 99311 UBAP2 Ubiquitin-associated protein 2 171127 + UBE2O Ubiquitin-conjugating 783378 enzyme E2 O UCK2 Isoform 1 of Uridine-cytidine 65671 + kinase 2 UQCC 34 kDa protein 872061 URB1 Nucleolar pre-ribosomal- 297241 associated protein 1 UTP20 Small subunit processome 4970 component 20 homolog VAMP7 Isoform 3 of Vesicle- 401804 associated membrane protein 7 WDR43 WD repeat-containing 55954 protein 43 WDR74 Isoform 1 of WD repeat- 18192 + containing protein 74 XPO5 Isoform 1 of Exportin-5 640703 XPOT Exportin-T 306290 YLPM1 YLP motif containing 1 165434

DEFINITIONS

The terms “subject” and “patient” as used herein refer to any single subject for which cancer detection, prognosis, staging or therapy is desired, including humans and non-human mammals, such as primate, bovine, ovine, canine, feline and rodent mammals. Also included are subjects involved in clinical research trials not showing any clinical sign of disease, or subjects involved in epidemiological studies, or subjects used as controls.

The terms “cancer detection” and “cancer diagnosis” and related grammatical terms, such as “detecting cancer” and “diagnosing cancer”, respectively, are used herein interchangeably to refer to any of determination of a subject's susceptibility to a malignant cancer disease; determination as to whether a subject is presently affected by a malignant cancer disease; determination of a subject's stage of cancer, determination of and monitoring the effect on the cancer in response to anti-cancer therapy.

The term “characteristics of a cancerous or pre-cancerous growth” as used herein refers to one or more molecular, physiological, histological, clinical or other properties which may be used to define the nature and behavior of the growth.

The terms “cancer”, “neoplasm”, “tumor”, “growth” and the like are used interchangeably herein to refer to cells which exhibit relatively autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In general, cells of interest for detection or treatment in the present application include pre-malignant (e.g., benign hyperplasic), malignant, metastatic, and non-metastatic cells.

The term “prognosis” as used herein refers to the expected or predicted outcome of a disease, such as a cancer, in a patient following diagnosis. A prognosis may predict the relative chance of disease progression, arrest or cure. A prognosis may be established on the basis of prognostic indicators specific for a particular disease. Prognostic indicators in cancer may include for example, the grade and stage of cancer at initial diagnosis, the genetic make-up of the patient, the presence and level of cancer-associated antigens in the tumor, and patient responsiveness to a particular therapy.

The terms “biological sample” and “test sample” as used herein encompass a variety of types of biological materials that can be used in the methods of the invention. The sample may be procured from the body of an individual or investigated without removal from the body of an individual. The term encompasses solid tissue samples, such as from biopsy specimens, tumors or tumor metastases, or tissue cultures or cells derived there from and the progeny thereof. The terms also encompass blood and other liquid samples of biological origin, such as nipple aspirate fluid, lymph node aspirate, mucosal fluid, cervical wash, lacrimal duct fluid, urine, saliva, pleural effusion and sputum. The terms encompass samples that have been manipulated in any way after their procurement, such as by lysis, treatment with reagents, solubilization, or enrichment for certain components. Also included are clinical samples, cells in cell culture, cell supernatants and cell lysates. It is to be explicitly understood that in accordance with the invention, a biological or test sample may be obtained i.e. removed, from the body of a subject, or accessed in vivo, for example by contacting with a specific reagent or apparatus.

As used herein, a biological or test sample that is “obtained from a subject”, means that the sample is removed from the body of the subject, and any subsequent analysis thereof may be performed outside the body for example under in vitro or ex vivo conditions. When however, a biological or test sample is “assessed in vivo” or “accessed in vivo” it means that the sample is maintained within the body of the subject and direct or indirect contact is established using any suitable means, such as via a reagent, composition, device or apparatus. Subsequent analysis of the accessed sample is performed under in vivo conditions, which can include conditions of local or general anaesthesia. Means of accessing and assessing a sample in vivo include for example, contacting the tissue or organ or region thereof of interest with a pharmaceutical composition, a swallowable endoscopy capsule or an endoscopy probe.

The term “a normal biological sample of the same type” as used herein refers to a non-diseased sample consisting of the same biological material or type of tissue and/or cells e.g. blood, colorectal tissue, as that of the test sample. The normal biological sample may be that from a single individual, including the subject in which cancer detection, prognosing or characterizing is performed, or from a group of individuals of known healthy status. Accordingly, the level of a protein in a normal biological sample may be obtained from a single determination or may advantageously represent a statistical average of multiple determinations, such as from a group of healthy individuals or from multiple healthy tissue sites in a single individual.

The term “a control sample” as used herein refers to the standard provided by either a normal i.e. non-diseased sample or group of samples, or a sample or group of samples corresponding to an established form, type, stage or grade of a disease, in particular a cancer disease. Accordingly, the level of a protein in a control sample may be obtained from a single determination or may advantageously represent a statistical average of multiple determinations, for example from a group of healthy individuals, or from a group of diseased individuals established to have the same form, type, stage or grade of a cancer disease.

The term “non-diseased tissue” as used herein refers to tissue which is determined to be free of a cancer disease, on the basis of any technique known in the art, for example, histological and immuno-histochemical investigation. It is to be understood that diseased and non-diseased tissue of the same tissue type may be present in close proximity in a individual patient.

The term “immunogenic fragment” as used herein in reference to a protein refers to a portion of the protein which is capable of inducing an immune response, such as antibody production, following administration to an individual.

As used herein, the terms “a protein associated with cancer”, “tumor associated protein”, “molecular marker” and the like, interchangeably refer to a protein that is present at relatively higher or lower levels in a cancer cell relative to a normal cell of the same type (e.g., as in protein associated with colon cancer).

As used herein the terms “nucleic acid encoding a protein,” encompass polynucleotides and polypeptides respectively having sequence similarity or sequence identity to the respective gene and gene products having the accession numbers of the particular protein referred to, of at least about 65%, preferably at least about 80%, more preferably at least about 85%, and can be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. Sequence similarity and sequence identity are calculated based on a reference sequence, which may be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. A reference sequence will usually be at least about 18 nt long, more usually at least about 30 nt long, and may extend to the complete sequence that is being compared. In general, percent sequence identity is calculated by counting the number of residue matches (e.g., nucleotide residue or amino acid residue) between the query and test sequence and dividing total number of matches by the number of residues of the individual sequences found in the region of strongest alignment, as is known in the art. Algorithms for computer-based sequence analysis are known in the art, such as BLAST (see, e.g., Altschul et al., J. Mol. Biol., 215:403-10 (1990)), particularly the Smith-Waterman homology search algorithm as implemented in MPSRCH program (Oxford Molecular).

The terms “protein” and “polypeptide” are used interchangeably herein to refer to polymeric forms of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.

As used herein, a “fusion protein” or “chimeric peptide” refers to a protein or polypeptide which comprises at least a portion of a first naturally occurring protein or polypeptide fused to least a portion of a second protein or polypeptide. For example, a fusion protein for targeting FAM62B may include a portion or all of an anti-FAM62B antibody fused with another peptide or polypeptide such as a protein label moiety or a cytotoxic protein.

The term “antibody” as used herein is used in the broadest sense and specifically encompasses monoclonal antibodies, humanized antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single chain antibodies and antibody fragments (e.g., F(ab′)₂, Fab′, Fab, Fv) so long as they bind specifically to a target antigen or epitope of interest.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries, as is known in the art, for example using techniques such as those described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

Furthermore, monoclonal antibodies specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see for example U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

The term “reagent suitable for detecting the level of a protein” as used herein refers to a reagent which either specifically binds the protein itself or is complementary to a nucleic acid molecule that is involved in expression of the protein. Particularly suitable examples of reagents which specifically bind to proteins are antibodies and chemical mimetics thereof having similar level of binding affinity and/or avidity. Nucleic acid reagents which are complementary to mRNA encoding a protein of interest are suitable for determining the level of the protein, typically by correlating the amount of mRNA detected with the corresponding level of protein product produced in a suitable translation system.

The terms “specifically interacts” and “specifically binds” are used herein interchangeably to refer to high avidity and/or high affinity binding of a reagent, such as an antibody to a specific polypeptide or epitope thereof, such as for example, an epitope of any of the proteins FAM62B, SLC1A5, RSL1D1, LYZ, RBM39 and TCOF1. Antibody binding to its epitope is stronger than binding of the same antibody to any other epitope, particularly those which may be present in molecules in association with, or in the same sample, as the specific polypeptide of interest. Antibodies which bind specifically to a polypeptide of interest may be capable of binding other polypeptides at a weak, yet detectable, level (e.g., 10% or less of the binding shown to the polypeptide of interest). Such weak binding, or background binding, is readily discernible from the specific antibody binding to the compound or polypeptide of interest, e.g., by use of appropriate controls.

The term “primary antibody” as used herein refers to an antibody which binds specifically to the target protein antigen in a biological or test sample. A primary antibody is generally the first antibody used in an immunoassay procedure. In some embodiments, the primary antibody is the only antibody used in an immunoassay procedure.

The term “secondary antibody” as used herein refers to an antibody which binds specifically to a primary antibody, thereby forming a bridge between the primary antibody and a subsequent reagent, if any. The secondary antibody is typically directed against the Fc portion of the immunoglobulin type of the primary antibody (e.g., anti-mouse Fc antibody).

The terms “polynucleotide” and “nucleic acid” are used interchangeably herein to refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxynucleotides, including but are not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. Further include are mRNA or cDNA that comprise intronic sequences (see, e.g., Niwa et al. (1999) Cell 99(7):691-702). The backbone of the polynucleotide can comprise sugars and phosphate groups (as typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidites and thus can be an oligodeoxynucleoside phosphoramidate or a mixed phosphoramidate-phosphodiester oligomer. Peyrottes et al. (1996) Nucl. Acids Res. 24:1841-1848; Chaturvedi et al. (1996) Nucl. Acids Res. 24:2318-2323. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars, and linking groups such as fluororibose and thioate, and nucleotide branches. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component, capping, substitution of one or more of naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides, or a solid support.

The term “complementary” as used herein refers to the broad concept of subunit sequence complementarity between two nucleic acids, e.g., two DNA molecules or a DNA/RNA hybrid. When a nucleotide position in both of the molecules is occupied by nucleotides normally capable of base pairing with each other, then the nucleic acids are considered to be complementary to each other at this position. Thus, two nucleic acids are complementary to each other when a substantial number (at least 50%) of corresponding positions in each of the molecules are occupied by nucleotides which normally base pair with each other (e.g., A:T and G:C nucleotide pairs).

The term “hybridization” as used herein encompasses any process by which a strand of nucleic acid joins with a complementary strand through base pairing (see for example, Coombs, Dictionary of Biotechnology, Stockton Press, New York N.Y. (1994)). Accordingly, a hybridization assay is a quantitative means of determining the extent of hybridization between a nucleic acid in a test sample, such as a tissue sample, and a nucleic acid probe corresponding to at least a fragment of a gene (DNA or RNA) encoding a protein of interest, such as a cancer associated protein. “Stringency” typically occurs in a range from about Tm −5° C. (5° C. below the Tm of the nucleic acid probe) to about 20° C. to 25° C. below Tm. As will be understood by those of skill in the art, a stringency hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences.

Amplification as carried out in the polymerase chain reaction technologies is described in Dieffenbach et al., PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y. (1995), and may be performed prior to or as part of a hybridization assay.

As used herein, the term “differentially expressed” generally refers to a polynucleotide and/or the corresponding protein that is expressed at levels in a test cell that differ significantly from levels in a reference or control cell, e.g., a cancer-associated protein as disclosed herein is found at levels at least about 50% to about 100% increased, generally at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, or at least about 30-fold or more increased in a cancerous cell when compared with a cell of the same type that is not cancerous. The comparison can be made between two tissues, for example, if one is using in situ hybridization or another assay method that allows some degree of discrimination among cell types in the tissue. The comparison may also be made between cells removed from their tissue source, or cells maintained in their native state in vivo. “Differential expression” refers to both quantitative, as well as qualitative, differences in the genes' temporal and/or cellular expression patterns among, for example, normal and neoplastic tumor cells, and/or among tumor cells which have undergone different tumor progression events.

The terms “correspond to” or “represents” as used in, for example, the phrase “polynucleotide corresponds to a differentially expressed gene” are used to refer to the relationship between a given polynucleotide and the gene from which the polynucleotide sequence is derived (e.g., a polynucleotide that is derived from a coding region of the gene, a splice variant of the gene, an exon, and the like) or to which the polynucleotide hybridizes to under stringent conditions.

The term “label” as used herein refers to a compound or composition which is conjugated or fused directly or indirectly to a reagent such as an antibody, a nucleic acid probe or a chemical agent and facilitates detection of the reagent to which it is conjugated or fused. The label may itself be detectable (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.

The term “mimetic” as used herein refers to any entity, including natural and synthesized inorganic or organic molecules, including recombinant molecules, that mimic the properties of the molecule of which it is a mimetic. Accordingly, a mimetic of a particular antibody has the same, similar or enhanced epitope binding properties of that antibody.

The term “gene” as used herein refers to any nucleic acid sequence or portion thereof with a functional role in encoding or transcribing a protein or regulating other gene expression. The gene may consist of all the nucleic acids responsible for encoding a functional protein or only a portion of the nucleic acids responsible for encoding or expressing a protein. The nucleic acid sequence may contain a genetic abnormality within exons, introns, initiation or termination regions, promoter sequences, other regulatory sequences or unique adjacent regions to the gene.

The singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the reagent” includes reference to one or more reagents and equivalents thereof known to those skilled in the art, and so forth.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Detection, Prognosis and Management of Colorectal Cancer

The present invention is based on the discovery that specific proteins, including for example, KIAA0152, NAMPT, PYCR1, GPX2, PRKDC, ALDH18A1, OCIAD2, GCS1, GMDS, ARF4, ARF5, LRPPRC, CTNNB1, ARF3, GCN1L1, BDH1, RPL9, UGCGL1, FAM3D and CCT4, CPT2, ARL1, PFKL, GOT2, AP1G1, STRBP, CLCA1, CYFIP1, COQ9, NDUFA9, ALDH7A1, HMGCS1, NNT, PRDX5, PCCB, COPZ1, BAX, ACAD9, UBXD8, HMGCS2, SLC25A3, SLC25A11, PDCD6, UCRC, DEFA6, DYNC1H1, HK1, CYFIP2, DCI, CISD1, FAM62B, S100A8, S100A9, MPO, MCM2, LTF, OLFM4, FERMT1, CEACAM5, SLC1A5, THBS1, NAT10, RSL1D, LMO7, LYZ and MCM3 are present at significantly higher levels in cancerous or pre-cancerous colorectal tissue as compared to normal tissue of the same cell type, even in the same individual.

This discovery serves as the basis for identification of cancerous tissue, as well as for staging and prognosing tumors, and development of therapeutic agents which target the particular cancer-associated markers. Detection of the markers disclosed herein and/or the genes encoding them, enables early diagnosis based on molecular changes leading to carcinogenesis and/or decision making in disease management. For example, a relatively increased level of a particular protein, such as FAM62B, compared to normal cells or tissues of the same type can be indicative of a poorer prognosis, and therefore warrant more aggressive therapy (e.g., chemotherapy following surgery). The correlation of tumor specific markers with response to treatment and outcome in patients can define prognostic indicators that allow the design of tailored therapy based on the molecular profile of the tumor. These therapies include antibody targeting, antagonists (e.g., small molecules), and gene therapy. Determining colon cancer-specific protein levels and comparison of a patient's profile with known levels in normal tissue and variants of the disease allows a determination of optimal treatment strategies. The marker expression pattern can also be used to better classify (i.e. stage and grade), and thus diagnose and treat different forms of cancer. Furthermore, a protein identified as being differentially or specifically expressed in, one type of cancer may also have implications for development or risk of development of other types of cancer.

Colorectal cancer is one of the most common neoplasms in humans and perhaps the most frequent form of hereditary neoplasia. Prevention and early detection are key factors in controlling and curing colorectal cancer. Colorectal cancer begins as polyps, which are small, benign growths of cells that form on the inner lining of the colon. Over a period of several years, some of these polyps accumulate additional mutations and become cancerous. Multiple familial colorectal cancer disorders have been identified, as follows: 1) Familial adenomatous polyposis (FAP); 2) Gardner's syndrome; 3) Hereditary nonpolyposis colon cancer (HNPCC); and 4) Familial colorectal cancer in Ashkenazi Jews.

Staging is a process used in the medical arts to describe how advanced the cancerous state is in a patient. While staging systems vary with the types of cancer, they generally involve the “TNM” system: “T” indicates the type of tumor, “N” indicates whether the cancer has metastasized to nearby lymph nodes; and “M” indicates whether the cancer has metastasized to other parts of the body. Generally, if a cancer is only detectable in the area of the primary lesion without having spread to any lymph nodes it is called Stage I. If it has spread only to the closest lymph nodes, it is called Stage II. In Stage III, the cancer has generally spread to the lymph nodes in near proximity to the site of the primary lesion. Cancers that have spread to a distant part of the body, such as the liver, bone, brain or other site, are Stage IV, the most advanced stage.

The grade of a cancer describes how closely a tumor resembles normal tissue of its same type. The microscopic appearance of a tumor is used to identify tumor grade based on parameters such as cell morphology, cellular organization, and other markers of differentiation. As a general rule, the grade of a tumor corresponds to its rate of growth or aggressiveness, with undifferentiated or high-grade tumors generally being more aggressive than well differentiated or low-grade tumors. The following guidelines are generally used for grading tumors: GX, Grade cannot be assessed; G1, Well differentiated; G2, Moderately well differentiated; G3, Poorly differentiated; G4, Undifferentiated.

Methods of detection, prognosis, and characterization as disclosed herein are based on levels of at least one, and preferably a panel or multiplicity of cancer-associated proteins, in comparison to levels of the same protein(s) in suitable non-cancerous or cancerous control samples. For example, a detection of cancer may be enabled by the detection of a level of one or more proteins of interest in a sample, such as FAM62b, that is increased at least by 50% or 100% or greater or, alternatively by 2-fold, 3-fold 5-fold, 10-fold, 30-fold, or greater, relative to a normal non-cancerous sample of the same tissue type. The normal non-cancerous sample may be from the same individual as the test sample or from one or more different individuals. Preferably, the level in the normal non-cancerous sample is a statistical average of multiple determinations, such as from a group of healthy individuals or from multiple healthy tissue sites in a single individual.

Similarly, prognosis and characterization of the cancer may be established on the levels of proteins of interest relative to reference standards established for particular grades, stages and forms of cancer. For example, detection of a level of a particular protein which is 1.5-fold compared to that of the control may be taken to indicate a relatively positive prognosis and/or relatively non-aggressive type of cancer, whereas detection of a level of the same protein of 10-fold or more compared to that of the control may be taken to indicate a poorer prognosis and/or a substantially aggressive type of cancer.

Assay Systems and Methods

The methods of the invention may be carried out using various assay systems and methods for detection of the level of the protein of interest in a test biological sample. Suitable systems include those employing an immunoassay, a nucleic acid hybridization assay, a binding assay, an array, a phage display library, or a combination thereof.

Immunoassays

Immunoassays for detecting levels of specific binding between an antibody and its target antigen are known in the art and include for example, radioimmunoassay, (RIA), fluorescent immunoassay, (FIA) enzyme-linked immunosorbant assay (ELISA), immunohistochemistry (IHC) and fluorescent activated cell sorting (FACS) (see, e.g., Harlow and Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1999)). In general, an immunoassay may be either direct or indirect. In a direct assay, the binding of antibody to the target antigen is determined directly using a labeled reagent, such as a fluorescent labeled or an enzyme-labeled primary antibody, which can be detected without further antibody interaction. In a typical indirect assay, unconjugated primary antibody binds to the antigen and then a labeled secondary antibody binds to the primary antibody. Signal amplification occurs because several secondary antibodies may react with different epitopes on the primary antibody. Alternately, both the primary and secondary antibodies may be unlabeled and labeled tertiary antibody is employed. Where the antibody (primary, secondary or tertiary) is conjugated to an enzymatic label, a chromagenic or fluorogenic substrate is added to provide detection of the antigen.

The primary antibody used for detection of the protein(s) of interest is stably associated with e.g., directly or indirectly bound to, the surface of a solid support, e.g. column, microtiter plate, beads, membrane, typically made of glass, plastic, polysaccharides, nylon or nitrocellulose. A multiplicity of antibody specificities for detecting a panel of tumor-associated proteins may be simultaneously bound to the same support, such as in an array.

The test sample is allowed to contact the support during a period of incubation, generally following blocking of non-specific binding sites with non-interfering proteins such as bovine serum albumin. After incubation with each reagent e.g. blocking agent, primary antibody, secondary antibody, the support is washed to remove non-bound components. Determination of suitable reagents, conditions for washing, incubation etc. is within the ability of one of average skill in the art.

Immunoassays can also be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of tumor associated antigen. In situ detection can be accomplished by removing a histological sample from a subject, and contacting the sample with a labeled antibody. The antibody is typically contacted with the sample by overlaying the labeled antibody onto the sample. Through the use of such a procedure, the presence of the tumor associated antigen can be determined and/or the distribution of the antigen in the histological sample can be examined. Those of ordinary skill in the art will readily appreciate that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

Detectable labels suitable for conjugation to antibodies and other binding reagents include radioisotopes, fluorescent labels, enzyme-substrate labels, chromogenic labels, chemiluminescent labels and colloidal gold particles.

Radioisotopes include for example, ³⁵S, ¹⁴C, ¹²⁵I, ³²P and ¹³¹I. Fluorescent labels include for example, fluorescent molecules fluorescein isothiocyanate (FITC), rhodamine, phycoerythrin (PE), phycocyanin, allophycocyanin, ortho-phthaldehyde, fluorescamine, peridinin-chlorophyll a (PerCP), Cy₃ (indocarbocyanine), Cy₅ (indodicarbocyanine), lanthanide phosphors, and the like.

Enzymatic labels include luciferases (e.g. firefly luciferase and bacterial luciferase), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Examples of enzyme-substrate combinations include, for example: horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor (e.g., orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidine hydrochloride (TMB)); alkaline phosphatase (AP) with para-nitrophenyl phosphate as chromogenic substrate; and β-D-galactosidase β-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate (e.g., 4-methylumbelliferyl-β-D-galactosidase).

A label may be indirectly conjugated with an antibody or other reagent, as is known in the art. For example, an antibody can be conjugated with biotin and any of the types of labels mentioned above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin, and thus, the label can be conjugated with the antibody in an indirect manner. In some cases, detectable labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

Detection of bound, labeled antibody can be carried out by standard colorimetric, radioactive, photometric and/or fluorescent detection means. For fluorescent labels, signal can be detected by, for example, a scanning confocal microscope in photon counting mode. Appropriate scanning devices are described by, for example, U.S. Pat. Nos. 5,578,832 and 5,631,734. For antibodies labeled with biotin, the reaction can be treated with the appropriate streptavidin-conjugate (e.g., streptavidin-horseradish peroxidase, streptavidin-alkaline phosphatase, streptavidin-luciferase, and the like) and then treated with the appropriate reagents for calorimetric or photometric detection. For radiolabeled antibody, signal can be detected using a scintillation counter, phosphoimager or similar device.

Arrays

Cancer associated proteins in a sample may be detected using an array-based binding assay system. Such an array-based system may incorporate an immunoassay as described above, or incorporate small molecule chemical entities which specifically interact with particular cancer associated proteins. In either case, the solid substrate used for the array comprises a plurality of binding reagents attached to the substrate, wherein each binding reagent has specificity for a different cancer associated protein. The protein panel or “set” for which the array is predetermined to specifically bind and detect is characteristic of a particular disease, or stage of form of a disease. The binding reagents, are immobilized onto the substrate surface, preferably in a spatially addressable manner. The binding reagents may be antibodies, antibody fragments or small molecule chemical entities.

The nature and geometry of the solid substrate will depend upon a variety of factors, including, among others, the type of array (e.g., one-dimensional, two-dimensional or three-dimensional). Generally, the surface can be composed of any material which will permit immobilization of the binding reagents and which will not substantially degrade under the conditions used in the applications of the array.

The solid substrate used for the array may be in the form of beads, particles or sheets, and may be permeable or impermeable, depending on the type of array, wherein the surface is coated with a suitable material enabling binding of the binding reagents at high affinity. For example, for linear or three-dimensional arrays the surface may be in the form of beads or particles, fibers (such as glass wool or other glass or plastic fibers) or glass or plastic capillary tubes. For two-dimensional arrays, the solid surface may be in the form of plastic, micromachined chips, membranes, slides, plates or sheets in which at least one surface is substantially flat, wherein these surfaces may comprise glass, plastic, silicon, low cross-linked and high cross-linked polystyrene, silica gel, polyamide, and the like.

Fluorescence tagged beads are also an addressable (liquid) array in which each bead is tagged with a different set of fluorescent colors and bound with an antibody; specific to an array is detected with devices such as fluorescence scanners for arrays or FACS for beads.

The arrays used for the present invention may be of any desired size. The upper and lower limits on the size of the array are determined solely by the practical considerations of resolution, size of molecules expressed at each address and the like.

Either a population of discrete proteins is employed to form the array, such that each address presents a different molecule, or a single or a few addresses are employed with a similar protein. In many applications, redundancies in the spots are desirable for the purposes of acting as internal controls.

Technologies for the deposition of droplets containing protein binding reagents onto a suitable solid surface are known in the art. An ink-jet printing technology for deposition of small droplets while avoiding overlap or splatter is disclosed in U.S. Pat. No. 5,449,754.

In order to conduct array-based binding assays, the test sample is allowed to contact the array comprising a coated surface containing the anchored binding reagents. Following contact, the array is optionally washed, typically under conditions such that any complexes formed will remain immobilized on the solid surface and unbound material will be removed.

The detection of complexes anchored on the solid surface can be accomplished in a number of ways. In some embodiments, the non-immobilized sample is pre-labeled, and the detection is directed to label immobilized on the surface indicating that complexes were formed. In other embodiments, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the previously non-immobilized sample (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody). In another embodiment, the immobilized molecules of the microarray are labeled, the array can be scanned or otherwise analyzed for detectable assay signal, and the signal from each labeled spot, or alternatively from all spots, quantified.

An important consideration is the presence of an amount of a label at each position within the array that is proportional to the amount of molecule immobilized at that particular spot. Thus, it is important that the efficiencies of the coupling reactions which are used to immobilize the labeled molecules are substantially similar.

Virtually any label that produces a detectable, quantifiable signal and that is capable of being attached to an immobilized binding reagent on a substrate can be used in conjunction with the array of the invention. Suitable labels include: radioisotopes, fluorophores, chromophores, chemiluminescent moieties, as described above.

Preferably, the position of the label will not interfere with interaction between a desired sample and the immobilized molecules and with the detection in case of an interaction between the desired sample and an immobilized molecule of the array. Suitable methods of making labeled molecules are well known in the art.

In the case where each spot in the array contains an amount of a label or “tracer” proportional to the amount of molecules immobilized at the particular spot, the signals obtained from the arrays of the invention can be normalized. As a consequence, signal intensities from spots within a single array, or across multiple arrays, can be directly compared. A normalized signal of a particular spot may be defined by (I_(t)−I₀)/I_(o), where I_(t) is the intensity of the signal of the spot after contacting with a sample of interest and I_(o) is the intensity of the background signal of the spot before contacting with a sample of interest.

Various methods and devices for detection and analysis of the array are known in the art. Practically, any imaging system that is capable of detecting with a resolution appropriate to the size of the array features can be utilized. For example, a method for screening an array of proteins for interactions with a fluid sample is disclosed in U.S. Pat. No. 6,475,809. Imaging apparatus may be selected, for example, from ScanArray 4000 (General Scanning), Biochip Imager (Hewlett Packard), GMS 418 Array Scanner (Genetic Microsystems), GeneTAC 1000 (Genomic Solutions), Chip Reader (Virtek). Phosphorimager systems are available for detecting radiolabels, e.g. Cyclone (Packard Instrument Co.) and BAS-5000 (Fujifilm).

Hybridization Assays

Hybridization assays generally comprise contacting a sample containing nucleic acids (target nucleic acids) with a nucleic acid probe capable of hybridizing to tumor associated antigen nucleic acids, under conditions such that hybridization can occur, and detecting or measuring any resulting hybridization.

Suitable hybridization assays include, for example, Northern blots, dot blots, RT-PCR, and quantitative PCR. Such procedures can be performed in situ directly for example, in tissue sections (e.g., fixed and/or frozen) of subject tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Tumor associated antigen nucleic acids can be used as probes and/or primers for such procedures (see e.g., Nuovo, PCR In Situ Hybridization: Protocols and Applications, Raven Press, NY (1992)).

Detection of tumor associated antigen nucleic acids typically involves contacting and incubating nucleic acids from a test sample with one or more labeled nucleic acids, (i.e. “probes”) under conditions favorable for the specific annealing of the nucleic acids to their complementary sequences. Typically, the lengths of the nucleic acid reagents are at least 15 to 30 nucleotides. After incubation, all non-annealed nucleic acids are removed. The presence of bound i.e. hybridized, nucleic acids from the sample, if any such molecules exist, is then detected. Using such a detection scheme, the nucleic acid from the tissue or cell type of interest can be immobilized, to a solid support such as a membrane, or a plastic surface such as that on a microtiter plate or polystyrene beads.

Nucleic acid arrays can be used to monitor the expression of tumor associated genes, such as, for example, those corresponding to KIAA0152, NAMPT, PYCR1, GPX2, PRKDC, ALDH18A1, OCIAD2, GCS1, GMDS, ARF4, ARF5, LRPPRC, CTNNB1, ARF3, GCN1L1, BDH1, RPL9, UGCGL1, FAM3D and CCT4, CPT2, ARL1, PFKL, GOT2, AP1G1, STRBP, CLCA1, CYFIP1, COQ9, NDUFA9, ALDH7A1, HMGCS1, NNT, PRDX5, PCCB, COPZ1, BAX, ACAD9, UBXD8, HMGCS2, SLC25A3, SLC25A11, PDCD6, UCRC, DEFA6, DYNC1H1, HK1, CYFIP2, DCI, CISD1, FAM62B, S100A8, S100A9, MPO, MCM2, LTF, OLFM4, FERMT1, CEACAM5, SLC1A5, THBS1, NAT10, RSL1D, LMO7, LYZ and/or MCM3. For detection of a multiplicity of genes encoding distinct cancer associated proteins, an array of polynucleotide probes may be contacted with a sample of target nucleic acids to produce a hybridization pattern. The binding of the target nucleic acids to one or more probes of the array is then detected to obtain a qualitative and/or quantitative profile of expression of the tumor associated antigen gene.

A variety of different arrays can be used and are known in the art. The polymeric or probe molecules of the arrays can be polynucleotides or hybridizing derivatives or analogs thereof, including: nucleic acids in which the phosphodiester linkage has been replaced with a substitute linkage, such as phosphorothioate, methylimino, methyl-phosphonate, phosphoramidate, guanidine, and the like; nucleic acids in which the ribose subunit has been substituted, for example, hexose phosphodiester; peptide nucleic acids; and the like. The length of the probes will generally range from about 10 to about 1000 nucleotides, typically, from about 15 to about 150 nucleotides in length, but also possibly from about 150 to about 1000 nucleotides in length. The probes can be single or double stranded, usually single stranded, and can be PCR fragments amplified from cDNA. The probe molecules on the surface of the substrates will typically correspond to at least one of the tumor associated antigen genes and be positioned on the array at known locations so that positive hybridization events can be correlated to expression of a particular gene in the physiological source from which the target nucleic acid sample is derived. Because of the manner in which the target nucleic acid sample is generated, the arrays of probes will generally have sequences that are complementary to the non-template strands of the gene to which they correspond.

The substrate for the array can be fabricated from a variety of materials, including plastics, ceramics, metals, gels, membranes, glasses, and the like. The arrays can be produced according to any convenient methodology, such as pre-forming the probes and then stably associating them with the surface of the support or growing the probes directly on the support. A number of different array configurations and methods for their production are known to those of skill in the art and disclosed in, for example, U.S. Pat. Nos. 5,445,934; 5,532,128; 5,556,752; 5,242,974; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327; 5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554,501; 5,561,071; 5,571,639; 5,593,839; 5,599,695; 5,624,711; 5,658,734; and 5,700,637.

The target nucleic acid is typically contacted with the array under conditions sufficient for hybridization of target nucleic acid to probe to occur. Suitable hybridization conditions are well known to those of skill in the art (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed., Cold Spring Harbor, Cold Spring Harbor, N.Y. (2001)).

The amount of tumor associated antigen nucleic acids in the sample can be quantitated (see, e.g., U.S. Pat. No. 6,004,755). For example, the target nucleic acids in the sample can be end-labeled in a manner such that each of the target nucleic acids in the sample produces a signal of the same specific activity. By generating the same specific activity is meant that each individual target polynucleotide in the sample being assayed is labeled in a manner such that the molecule is capable of providing the same signal (e.g., the same intensity of signal) as every other labeled target in the sample. Each of the target nucleic acids generates a signal of the same specific activity because the number of labeled nucleotide bases in each of the target molecules is either identical or substantially the same.

The label is capable of providing a detectable signal, either directly or through interaction with one or more additional members of a signal producing system. Suitable detectable labels include radioactive, fluorescent and enzymatic labels as described above.

In some applications, it is desired to analyze populations of target nucleic acids from two or more samples. Such samples can be differentially labeled. Alternatively, target nucleic acids from different samples are separately contacted to identical probe arrays under conditions of hybridization, typically stringent hybridization conditions, such that labeled nucleic acids hybridize to their complementary probes on the substrate surface, and the target nucleic acids bound to the array separately detected. A set of standard nucleic acid molecules can optionally be used. For example, the standard nucleic acids can be provided by reverse transcribing standard RNA.

Following hybridization, a washing step is usually employed to remove non-specifically bound nucleic acid from the support surface, generating a pattern of hybridized nucleic acid on the substrate surface. Various wash solutions and protocols for their use are known to those of skill in the art.

Where the label on the target nucleic acid is not directly detectable, the array can be contacted with the other member(s) of the signal producing system that is being employed. For example, where the label on the target is biotin, the array can be contacted with streptavidin-fluorophore conjugate under conditions sufficient for binding between the specific binding member pairs to occur. Following contact, any unbound members of the signal producing system will then be removed (e.g., by washing).

The resultant hybridization pattern(s) of target nucleic acids bound to the array can be visualized or detected in a variety of ways, with the particular manner of detection being chosen based on the particular label of the nucleic acid. For example, detection means can include scintillation counting, autoradiography, fluorescence measurement, colorimetric measurement, light emission measurement, and the like.

Prior to detection or visualization, the array of hybridized target/probe complexes can be optionally treated with an endonuclease, for example, mung bean nuclease, S1 nuclease, and the like. The endonuclease degrades single stranded, but not double stranded DNA.

Following detection or visualization, the hybridization pattern can be used to determine qualitative and/or quantitative information about the expression of tumor associated antigen genes. The hybridization patterns of different samples can be compared with each other, or with a control sample, to identify differences between the patterns. The hybridization arrays can also be used to identify differential gene expression, in the analysis of diseased and normal tissue.

Antibody Production

Antibodies directed against cancer associated proteins include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, antibody fragments (e.g., Fab, Fab′, F(ab′)₂, Fv, or hypervariable regions), bi-specific antibodies and humanized antibodies, methods of production of which are known in the art.

For the production of polyclonal antibodies, a host animal (e.g., rabbits, mice, rats, sheep, goats, and the like) can be immunized by injection with a tumor associated antigen, fragment, derivative or analog. Various adjuvants can be used to increase the immunological response, depending on the host species. Such adjuvants include, for example, Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and other adjuvants, such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

Techniques for preparation of monoclonal antibodies include the original Kohler and Milstein hybridoma technique (see, e.g., Nature 256:495 97 (1975)), the trioma technique (see, e.g., Hagiwara and Yuasa, Hum. Antibodies Hybridomas 4:15 19 (1993); Hering et al., Biomed. Biochim. Acta 47:211 16 (1988)), the human B-cell hybridoma technique (see, e.g., Kozbor et al., Immunology Today 4:72 (1983)), and the EBV-hybridoma technique (see, e.g., Cole et al., In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77 96 (1985)). Human antibodies can be obtained using human hybridomas (see, e.g., Cote et al., Proc. Natl. Acad. Sci. USA 80:2026 30 (1983)) or by transforming human B cells with EBV virus in vitro (see, e.g., Cole et al., supra).

Chimeric antibodies are typically prepared by splicing the genes (of one species) for an antibody molecule specific for tumor associated antigen together with genes from another species of antibody molecule of appropriate biological activity. It can be desirable to transfer the antigen binding regions (e.g., Fab′, F(ab′)₂, Fab, Fv, or hypervariable regions) of antibodies from one species into the framework of an antibody from another species by recombinant DNA techniques to produce a chimeric molecule. Methods for producing such molecules are described in, for example, U.S. Pat. Nos. 4,816,567; 4,816,397; 5,693,762, and 5,712,120. A human monoclonal antibody or portion(s) thereof can be identified by screening a human B-cell cDNA library for nucleic acid molecules that encode antibodies that specifically bind to a tumor associated antigen according to the method generally set forth by Huse et al. (Science 246:1275 81 (1989)). The nucleic acid molecule can then be cloned and amplified to obtain sequences that encode the antibody (or antigen-binding domain) of the desired specificity. Phage display technology offers another technique for selecting antibodies that bind to tumor associated antigens, fragments, derivatives or analogs thereof (see, e.g., International Patent Publications WO 91/17271 and WO 92/01047; Huse et al., supra.)

Techniques for the production of single chain antibodies are described for example in U.S. Pat. Nos. 4,946,778 and 5,969,108. A Fab expression library (see, e.g., Huse et al., supra) allows rapid and easy identification of monoclonal Fab fragments with the desired specificity for tumor associated antigens, fragments, derivatives, or analogs thereof.

F(ab′)₂ antibody fragments can be produced by pepsin digestion of an antibody molecule. Fab′ fragments can be generated by reducing the disulfide bridges of a F(ab′)₂ fragment, Fab and Fv fragments can be generated by treating an antibody molecule with papain and a reducing agent. Recombinant Fv fragments can also be produced in eukaryotic cells using, for example, as described in U.S. Pat. No. 5,965,405.

Bi-specific antibodies can be monoclonal antibodies that have binding specificities for at least two different antigens. For example, one of the binding specificities can be for a tumor associated antigen and the other one is for any other antigen. Alternatively, one specificity is for a first tumor associated antigen, while the other specificity is for a second, different tumor associated antigen. Methods for making bi-specific antibodies may be based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (see, e.g., Milstein and Cuello, Nature 305:537 39 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas produce a potential mixture of different antibody molecules, some of which have the desired bi-specific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps.

Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion typically is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. The first heavy-chain constant region (CH1) containing the site necessary for light-chain binding is usually present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bi-specific antibodies see, for example, Suresh et al (Methods in Enzymology 121:210 (1986)).

To generate a phage antibody library, a cDNA library is first obtained from mRNA which is isolated from cells, e.g., the hybridoma, which expresses the desired protein to be expressed on the phage surface, e.g., the desired antibody. cDNA copies of the mRNA are produced using reverse transcriptase. cDNA which specifies immunoglobulin fragments are obtained by PCR and the resulting DNA is cloned into a suitable bacteriophage vector to generate a bacteriophage DNA library comprising DNA specifying immunoglobulin genes. The procedures for making a bacteriophage library comprising heterologous DNA are well known in the art and are described, for example, in Sambrook et al., supra.

Pharmaceutical Compositions and Agents

A pharmaceutical or antigen composition according to the invention comprises at least one protein selected from the group consisting of KIAA0152, NAMPT, PYCR1, GPX2, PRKDC, ALDH18A1, OCIAD2, GCS1, GMDS, ARF4, ARF5, LRPPRC, CTNNB1, ARF3, GCN1L1, BDH1, RPL9, UGCGL1, FAM3D, CCT4, CPT2, ARL1, PFKL, GOT2, AP1G1, STRBP, CLCA1, CYFIP1, COQ9, NDUFA9, ALDH7A1, HMGCS1, NNT, PRDX5, PCCB, COPZ1, BAX, ACAD9, UBXD8, HMGCS2, SLC25A3, SLC25A11, PDCD6, UCRC, DEFA6, DYNC1H1, HK1, CYFIP2, DCI, CISD1, LYZ, LOC442497;SLC3A2, DMBT1, NUCB1, GGH, AGR3, TM9SF2, SYK, GCA, HDLBP, C1QBP and CLIC1, or an immunogenic fragment thereof.

The composition may be used in a therapeutic method for preventing or treating cancer, wherein an effective amount of the composition is administered to a subject in need thereof. A subject in need thereof may be for example, an individual in which colorectal polyps have been identified, and/or has been determined to have other risk factors for development of colorectal cancer. Accordingly, a method of preventing colorectal cancer may comprise administering an effective amount of a pharmaceutical composition comprising at least one protein highly expressed in polyps, for example any of CPT2, ARL1, PFKL, GOT2, AP1G1, STRBP, CLCA1, CYFIP1, COQ9, NDUFA9, ALDH7A1, HMGCS1, NNT, PRDX5, PCCB, COPZ1, BAX, ACAD9, UBXD8, HMGCS2, SLC25A3, SLC25A11, PDCD6, UCRC, DEFA6, DYNC1H1, HK1, CYFIP2, DCI and CISD1.

Further provided are pharmaceutical compositions comprising one or more reagents suitable for detecting the level of at least one protein selected from the group consisting of KIAA0152, NAMPT, PYCR1, GPX2, PRKDC, ALDH18A1, OCIAD2, GCS1, GMDS, ARF4, ARF5, LRPPRC, CTNNB1, ARF3, GCN1L1, BDH1, RPL9, UGCGL1, FAM3D, CCT4, CPT2, ARL1, PFKL, GOT2, AP1G1, STRBP, CLCA1, CYFIP1, COQ9, NDUFA9, ALDH7A1, HMGCS1, NNT, PRDX5, PCCB, COPZ1, BAX, ACAD9, UBXD8, HMGCS2, SLC25A3, SLC25A11, PDCD6, UCRC, DEFA6, DYNC1H1, HK1, CYFIP2, DCI, CISD1, LYZ, LOC442497;SLC3A2, DMBT1, NUCB1, UGH, AGR3, TM9SF2, SYK, GCA, HDLBP, C1QBP and CLIC1.

As hereinbefore described, such reagents may comprise labeled antibodies, antibody mimetics or nucleic acids as the active ingredients. Advantageously such reagents are provided as a “cocktail” tailored to detect in vivo a particular panel of cancer associated proteins, or genes expressing same, Following administration, the localization of such reagents to specific body and organ regions may be detected by means of an external monitoring apparatus or system appropriate for detection and quantitation of the specific label incorporated into the reagents.

In yet other embodiments, pharmaceutical agents, in particular antibodies and an antibody mimetics, are provided, which may be used for targeted therapy of cancers expressing particular cancer associated proteins. Such, agents may advantageously incorporate a cytotoxic moiety, such as a plant toxin, a bacterial toxin, a radioactive atom or a chemotherapeutic agent. Further, the pharmaceutical agent may be in the form of a chemical conjugate or a fusion protein and provided in a suitably formulated pharmaceutical composition.

Plant toxins include for example, ricin, abrin, pokeweed antiviral protein, saporin, gelonin, and derivatives thereof. Bacterial toxins include for example, Pseudomonas exotoxin, diphtheria toxin and derivatives thereof, iodine-125 and radon. Radioactive atoms include for example, radium, cesium-137, iridium-192, americium-241 and gold-198.

Chemotherapeutic agents include, without limitation, alkylating agents, for example cyclophosphamide; nitrosoureas, for example carmustine and lomustine; antimetabolites, for example 5-fluorouracil, capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine and pemetrexed; anthracyclines, for example daunorubicin, doxorubicin respinomycin D and idarubicin; topoisomerase inhibitors, for example topotecan, irinotecan, etoposide and teniposide; mitotic inhibitors, for example paclitaxel, docetaxel, etoposide, vinblastine and vincristine; platinum based drugs, for example cisplatin, carboplatin and oxaliplatin; steroids for example hydrocortisone, dexamethasone, methylprednisolone and prednisolone; and anti-angiogenic agents, for example bevacizumab, thalidomide, dopamine and tetrathiomolybdate.

The pharmaceutical composition can be used to administer diagnostic reagents designed to detect levels of proteins disclosed herein, in particular KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20), including antibodies, antibody mimetics or nucleic acids, or therapeutic reagents designed to exert a cytotoxic affect on their cancer targets. Therapeutic compositions for targeting tumors may include a reagent which specifically interacts with a protein selected from NAMPT, PYCR1, GPX2, PRKDC, ALDH18A1, OCIAD2, GCS1, GMDS, ARF4, ARF5, LRPPRC, CTNNB1, ARF3, GCN1L1, BDH1, RPL9, KIAA0152, UGCGL1, FAM3D, CCT4, LYZ, LOC442497;SLC3A2, DMBT1, NUCB1, GGH, AGR3, TM9SF2, SYK, GCA, HDLBP, C1QBP, KIAA0152 and CLIC1.

The pharmaceutical composition may be prepared by suspending the desired reagent in any pharmaceutically acceptable carrier, for example, HEPES buffered saline at a pH of about 7.8. Other pharmaceutically acceptable carriers which are useful include, but are not limited to, glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

The pharmaceutical compositions may be prepared in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents. Sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.

Pharmaceutical compositions that are useful in the methods of the invention may be administered, prepared, packaged, and/or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.

As used herein, the term “physiologically acceptable” ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

A formulation of a pharmaceutical composition of the invention suitable for oral administration may be prepared in the form of a discrete solid dose unit including, but not limited to, a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined amount of the active ingredient. Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, or an emulsion. As used herein, an “oily” liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water.

As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the invention are known in the art.

Kits

The invention also includes kits for detecting, diagnosing, prognosing or staging a cancer or a tumor in a mammal. The cancer or tumor can be of any of the types described herein, and is preferably colorectal cancer. The kit comprises a container or a sample tube, or the like, for storing a sample of a cell, a population of cells, a tissue or a body fluid obtained from the mammal.

The kit also comprises one or more detection reagents selected from: an antibody or antibody mimetic which specifically bind with a cancer associated protein disclosed herein; a nucleic acid such as an oligonucleotide which specifically binds a nucleic acid (such as mRNA) encoding said cancer associated protein, and a PCR primer pair specific for a nucleic acid encoding said cancer associated protein. These detection reagents are as described herein. The kit comprises the one or more detection reagents in an amount effective to permit detection of the protein(s) of interest or a corresponding nucleic acid in the sample. Detection of the proteins or the nucleic acids is accomplished using any of the methods described herein or known to a skilled artisan for detecting a specific protein or specific nucleic acid molecule within a biological sample.

Reagents in the kit may be directed to a protein selected from KIAA0152, NAMPT, PYCR1, GPX2, PRKDC, ALDH18A1, OCIAD2, GCS1, GMDS, ARF4, ARF5, LRPPRC, CTNNB1, ARF3, GCN1L1, BDH1, RPL9, UGCGL1, FAM3D, CCT4, CPT2, ARL1, PFKL, GOT2, AP1G1, STRBP, CLCA1, CYFIP1, COQ9, NDUFA9, ALDH7A1, HMGCS1, NNT, PRDX5, PCCB, COPZ1, BAX, ACAD9, UBXD8, HMGCS2, SLC25A3, SLC25A11, PDCD6, UCRC, DEFA6, DYNC1H1, HK1, CYFIP2, DCI and CISD1. The kit may be intended for detecting pre-cancerous or early stage colorectal cancer and include reagents detecting any protein selected from the group consisting of CPT2, ARL1, PFKL, GOT2, AP1G1, STRBP, CLCA1, CYFIP1, COQ9, NDUFA9, ALDH7A1, HMGCS1, NNT, PRDX5, PCCB, COPZ1, BAX, ACAD9, UBXD8, HMGCS2, SLC25A3, SLC25A11, PDCD6, UCRC, DEFA6, DYNC1H1, HK1, CYFIP2, DCI and CISD1. In particular embodiments, the kit comprises a reagent suitable for detecting KIAA0152.

The kit also comprises at least one control biological sample, such as from non-diseased tissue, for comparison to a biological sample obtained from a subject under investigation. Control biological samples which correspond to samples from a cancer of a known stage or having a known prognosis, may also be included.

The kit also comprises an instructional material which directs the use of the reagents and the samples for the determining the amount and the location of the proteins or the nucleic acids in one or more cells of the sample. The instructional material also directs the correlation of the amount and the location of the protein or the nucleic acid in the cells of the sample with the diagnosis, prognosis and/or stage of a cancer or a tumor in the mammal.

As used herein, an “instructional material” includes a publication, a recording, a diagram, or any other medium of expression which directs or dictates the use of the components of a kit for performing the function of a method of the invention described herein. The instructional material of the kit of the invention may, for example, be affixed to a container which contains the composition or be shipped together with a container which contains the composition.

The following examples are presented in order to more fully illustrate certain embodiments of the invention. They should in no way, however, be construed as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES

The following methods were used in the Examples.

Protein Extraction from Frozen Tissues

Surplus snap-frozen tumor and margin non-tumoral tissue were collected from patients undergoing surgery for colorectal cancer at either of two university teaching hospitals in Israel. Around 30 mg of frozen tissue were extracted from every tissue sample by mixing the tissue sample with in 0.5 ml of 8 M urea, 400 mM ammonium bicarbonate, and homogenized by high speed tissue homogenizer (Omni-TH) for 1 min, activated at full speed.

Identification of Proteins

1) Gel-slicing method. About 50 μg of the proteins extracted from the tumor tissues or the healthy tissues were resolved by 10% of SDS-PAGE. Each gel lane was subsequently stained with Coomassie blue. The stained gel lanes were cut into 12 slices. The gel pieces were de-stained by extensive washing with acetonitrile and ammonium bicarbonate. The proteins in each gel slice were proteolyzed inside the stained gel: stained slices were reduced with 10 mM DTT, incubated at 60° C. for 30 min, alkylated with 10 mM iodoacetamide, at room temperature for 30 min and digested with trypsin overnight at 37° C., using modified trypsin (Promega) at a 1:100 enzyme-to-substrate ratio. The tryptic peptides were analyzed by AC-MS/MS using the OrbitrapXL mass spectrometer (Thermo-Fisher) fitted with a capillary HPLC (Eksigent). The peptides were resolved on homemade capillary columns (75 micron ID) packed with reversed phase 3.5 micron beads Reprosil C18-Aqua, using a method described by (Ishihama, Rappsilber et al. 2002). The HPLC separations of the peptides were at flow rates of about 250 nanoliters per minute during 2 hrs and with 7-40% gradients of acetonitrile in the presence of 0.1% formic acid. The capillary columns were connected on line to the Orbitrap mass spectrometer through an electrospray interface. The mass spectrometer was operated in a data-dependent mode where the masses of the eluting peptides were measured at high accuracy in the Orbitrap part of the machine and the seven most intense masses, detected at each full MS spectra whose charge states were determined to be double and triple, were selected for fragmentation by CID in the linear trap at the subsequent seven CID fragmentations.

2) Multidimensional chromatography coupled with mass spectrometry. 50 μg of total protein extracts mixed with 100 μg in 8 M urea, 100 mM ammonium bicarbonate were treated for blocking all the sulfhydryls by first reducing disulfides by addition of 10 mM DTT, incubation at 60° C. for 30 min. The free disulfides were next blocked by carboxymethylation using 10 mM iodoacetamide and incubation at room temperature for 30 min. The denatured and carboxymethylated protein mixtures were diluted three-fold with water to reduce urea concentration to about 2M followed by digestion in solution, overnight at 37° C. using modified trypsin (Promega) at a 1:100 enzyme-to-substrate ratio. The resulting peptides from the trypsinized proteins were desalted with reversed-phase with a C18 tip disposable micro-columns (Harvard), eluted with 90% acetonitrile, dried and dissolved in 0.1% formic acid. The resulting peptides were resolved by multi-dimensional chromatography with on-line first dimension of strong cation exchange (SCX) chromatography 0.3×5 mm columns (LC Packings) using ten salt steps of 20, 40, 60, 80, 100, 120, 160, 200, 300 and 500 mM ammonium acetate in 5% acetonitrile with 0.1% acetic acid. The peptides from each increased salt elution from the SCX columns were transferred on-line to a C18 trap column (0.3×5 mm, LC-Packings), which was connected on-line to a Reprosil C18 homemade capillary columns (75 micron ID), resolved by 7-40% acetonitrile gradients, during 2 hrs, in the presence of 0.1% formic acid as described before for the gel-slicing method.

Resolving peptides by two-dimensional capillary chromatography has been described (Link, Eng et al. 1999), reviewed in (Link 2002). The mass spectrometry analysis was performed on-line as described above using data-dependent LC-MS/MS analysis with full MS in the Orbitrap and subsequent seven dependent ion-trap CID spectra of the most abundant doubly and triply charged peptides, detected in the full MS.

3) Isotope labeling peptides to enable quantitative analysis. Labeling tryptic peptides with light or heavy stable isotope reagents may rely on commercial reagents, reviewed in (Ong and Mann 2005; Regnier and Julka 2006). To facilitate the accurate comparison of the relative amounts of each of the proteins in the different samples, the mixture of peptides produced by the trypsinization of the entire in-solution protein proteolysis were covalently modified with light and heavy stable-isotope reagents. Reductive dimethylation labeling was done with heavy and light formaldehyde as described by (Hsu, Huang et al. 2003).

50 ug of proteolytic peptides were covalently modified with stable-isotope labeled (heavy and light formaldehyde) reagents (reductive dimethylation). The labeled peptides were resolved by multi-dimensional chromatography with on-line SCX column as described above.

Stable isotope labeling was also performed by iTRAQ (Ross, Huang et al. 2004) using a labeling kit that was purchased from Applied Biosystems and labeling was done according to the manufacturer's protocol. The labeled peptides were resolved by multi-dimensional chromatography with on-line SCX column as described above.

Bioinformatics

The mass spectrometry data of both the tryptic peptides obtained from proteolysis in the gel slices mentioned above and the tryptic peptides resolved by multidimensional chromatography were clustered and analyzed using the Pep-Miner software tool (Beer, Barnea et al. 2004). The search against the human part of the IPI database was done by using multiple search engines: Pep-Miner (Beer, Barnea et al. 2004), Mascot (Perkins, Pappin et al. 1999) and Sequest (Eng, McCormack et al. 1994). Both Mascot and Sequest were run together using the Protein Discoverer software tool (Thermo-Fisher).

Peptides were selected according to the following critera: 1) Mascot: ionScore>identityHigh and expValue<0.05 and deltaScore=0; 2) Sequest: ((xCorr>2 and chg</=2) or (xCorr>2.5 and chg>/=3)) and probability>15 and deltaScore=0 A peptide was used for the analysis if it was identified with the above criteria at least once for Sequest and once for Mascot, although not necessarily both in the same scan or run or patient.

Example 1 Clinical Characteristics of Patients Undergoing Surgical Gastrointestinal Re-Sectioning

The entire protein repertoires of healthy and diseased gastrointestinal tissues were analyzed from samples obtained from greater than 50 patients undergoing surgery for colorectal cancer.

The clinical characteristics of the patients studied are provided in Table 2.

TABLE 2 Patient Age at No. Diagnosis Gender Appearance Diagnosis TNM Stage Grade 201 78 M primary Adeno- T3N1M0 III high carcinoma 202 81 F primary Mucinous T3N1 III low adeno- carcinoma 203 85 M primary Adeno- T3N0M0 II high carcinoma 205 71 F primary Adeno- T3N1M0 III high carcinoma 207 81 F primary Adeno- T3N2 III high carcinoma 208 78 M primary Adeno- T3N2M1 IV high carcinoma 209 71 M primary Adeno- T3N0M1 II high carcinoma 211 57 F primary Adeno- T2N0M0 I intermed carcinoma 212 50 F primary Adeno- T3N1M0 III high carcinoma 214 54 F primary Adeno- T3N1M0 III high carcinoma 217 77 F Local Adeno- T3N0M0 II high recurrance carcinoma 218 79 M primary Adeno- T3N2M1 IV low (T + P + N) carcinoma 219 60 M primary Adeno- T3N1M0 III high carcinoma 220 73 M primary Adeno- T3N2M1 IV high carcinoma Patient Age at Type of Tumor No. Diagnosis Gender surgery Diagnosis location Stage Grade J1 (T + N) 61 F Lt colectomy Adeno- left colon IV intermed carcinoma J2 (T + N) 67 M Lt colectomy Adeno- left colon II intermed carcinoma J3 (T + N) 70 M Rt colectomy Adeno- right colon II intermed carcinoma J4 (T + N) 83 F Sigmoidectomy Adeno- left colon II intermed carcinoma J5 (T + N) 41 M Lt colectomy Adeno- left colon I intermed carcinoma J6 (T + N) 67 M Lt colectomy Adeno- left colon IV intermed carcinoma J7 (T + N) 95 M Rt colectomy Adeno- right colon III intermed carcinoma (T3N1) J8 (T + N) 72 F Rt colectomy Adeno- right + left III high carcinoma colon T3N4 J9 (T + N) 56 F Rt colectomy Adeno- right colon II intermed carcinoma T4N0M0 J10 64 M Anterior Adeno- rectum II low (T + N) Resection carcinoma T4N0Mx of rectum J11 61 F Rt colectomy Adeno- right colon I low (T + N) carcinoma J12 61 M colectomy Adeno- rectum II intermed (T + N) carcinoma J13 60 M Rt colectomy Adeno- right colon I intermed (T + N) carcinoma J14 53 F colectomy Adeno- left colon II intermed (T + N) carcinoma J15 44 M Lt colectomy Colonic left colon (P + N) polyps J16 (P + N + 64 M Rt colectomy Tubulo- right colon Pbig) villus J17 81 M Rt colectomy Adeno- right colon II intermed (T + N) carcinoma J18 58 F Rt colectomy Adeno- right colon I intermed (P + N) carcinoma in TVA J19 72 M Rt colectomy Adeno- right colon II intermed (T + N + P) carcinoma J20 60 F Rt colectomy Adeno- left colon II intermed (T + N) carcinoma J21 29 F Rt colectomy Adeno- right colon IV low (T + N) carcinoma J22 70 M Rt colectomy Adeno- right colon II high (T + N) carcinoma J23 92 F Rt colectomy Adeno- right colon III intermed (T + N) carcinoma J24 75 M Rt colectomy Villo- right colon (P + N) tubulos adenoma J25 47 M Rt colectomy Adeno- Rectac- III intermed (T + N) carcinoma toigmoid J26 77 M Rt colectomy Adeno- right colon III intermed (T + N) carcinoma J27 77 M Lt colectomy Adeno- left colon II intermed (T + N) carcinoma J28 77 M ext Rt Villo- right colon high (Pbig + N + colectomy tubulos P) adenoma J29 78 M Lt colectomy Adeno- left colon II intermed (T + N) carcinoma J30 67 F Lt colectomy Adeno- right colon III intermed (T + N) carcinoma J31 83 M Sigmoidectomy Adeno- right colon II low (T + N) carcinoma J32 65 F Anterior Adeno- right colon II intermed (T + N) Resaction carcinoma J33 83 M Rt colectomy Adeno- right colon II intermed (N + P1 − 5) carcinoma J34 82 M Rt colectomy Adeno- right colon II intermed- (T + N) carcinoma high J35 81 M Gastrectomy Adeno- stomach III high (T + N) carcinoma J36 62 M Resaction Adeno- recto III intermed (T + N) carcinoma signuoid J37 55 M Rt colectomy Adeno- III intermed- (T + N) carcinoma high J38 80 M Rt colectomy Adeno- right colon III intermed- (T + N + P1 − carcinoma low P2) J39 (T + P) 62 M Rt colectomy Villo- right colon I high tubulos adenoma J40 52 M Anterior Adeno- Sigmoid III intermed- (T + N) Resection carcinoma low J41 87 M Rt colectomy Adeno- right colon III intermed- (T + N + P1 + carcinoma low P2) J42 87 M Rt colectomy Adeno- I intermed (T + N + P1 + carcinoma P2) T, tumor; N, normal; P, polyp; high grade = poorly differentiated; low grade = well differentiated; intermediate grade = moderately well differentiated.

Example 2 Identification of a Group of Protein Markers that are Found in Polyps and in Advanced Stages of Colorectal Cancer

Table 3 lists proteins that were observed to be highly expressed in polyps, as well as in early and advanced stages of various colorectal cancers. As shown in Table 3 and in FIG. 2, in a large number of patients, the expression level of these proteins in polyps or cancerous tissue was at least 3 times greater than the expression level in healthy tissue from the same patient. Similarly, few if any, of the patients exhibited decreased expression levels of the same proteins in polyps or cancerous tissue as compared to the corresponding level in healthy tissue from the same patient. That is, a ratio of protein expression (polyps/tumor vs healthy tissue from the same patient), that was less than 1:3, was rarely observed. Accordingly, a diagnostic array of reagents directed to detection of at least some of the proteins in this group may be used as a general screening test for colorectal cancers.

TABLE 3 IPI Acc. No. nPeptides Protein nBigT nSmallT nBigP nSmallP IPI00022649 18 SLC12A2 Isoform 1 of 14 2 16 0 Solute carrier family 12 member 2 IPI00017526 3 S100P Protein S100-P 27 0 15 0 IPI00022255 16 OLFM4 Olfactomedin-4 21 2 15 0 precursor IPI00024095 16 ANXA3 Annexin A3 19 1 15 0 IPI00555902 6 OCIAD2 Isoform 1 of 15 0 15 0 OCIA domain- containing protein 2 IPI00376503 7 PYCR1 pyrroline-5- 20 0 14 0 carboxylate reductase 1 isoform 2 IPI00298176 7 GPX2 Glutathione 18 0 14 0 peroxidase 2 IPI00296337 55 PRKDC Isoform 1 of 16 2 14 0 DNA-dependent protein kinase catalytic subunit IPI00215919 9 ARF5 ADP-ribosylation 11 2 14 0 factor 5 IPI00619903 21 UGCGL1 UDP- 9 2 14 0 glucose:glycoprotein glucosyltransferase 1 precursor IPI00018873 18 NAMPT Isoform 1 of 17 2 13 0 Nicotinamide phosphoribosyltransferase IPI00008982 16 ALDH18A1 Isoform 16 2 13 0 Long of Delta-1- pyrroline-5-carboxylate synthetase IPI00328170 6 GCS1 Mannosyl- 15 0 13 0 oligosaccharide glucosidase IPI00216057 8 SORD Sorbitol 14 2 13 0 dehydrogenase IPI00030207 16 GMDS GDP-mannose 13 2 13 0 4,6 dehydratase IPI00215918 10 ARF4 ADP-ribosylation 12 2 13 0 factor 4 IPI00783271 45 LRPPRC Leucine-rich 11 3 13 0 PPR motif-containing protein, mitochondrial precursor IPI00017292 23 CTNNB1 Isoform 1 of 11 2 13 0 Catenin beta-1 IPI00215917 10 ARF3 ADP-ribosylation 11 2 13 0 factor 3 IPI00001159 18 GCN1L1 Translational 11 1 13 0 activator GCN1 IPI00025341 10 BDH1 D-beta- 10 4 13 0 hydroxybutyrate dehydrogenase, mitochondrial precursor IPI00031691 9 RPL9 60S ribosomal 10 1 13 0 protein L9 IPI00029046 7 KIAA0152 10 0 13 0 Uncharacterized protein KIAA0152 precursor IPI00060143 5 FAM3D Protein 9 4 13 0 FAM3D precursor IPI00302927 18 CCT4 T-complex 8 1 13 0 protein 1 subunit delta IPI00643623 6 LCN2 Lipocalin 2 23 0 12 0 IPI00217223 13 PAICS Multifunctional 17 2 12 0 protein ADE2 IPI00012501 4 REG4 Isoform 1 of 16 0 12 0 Regenerating islet- derived protein 4 precursor IPI00793443 14 IPO5 RAN binding 15 1 12 0 protein 5 IPI00025273 15 GART Isoform Long of 15 1 12 0 Trifunctional purine biosynthetic protein adenosine-3 IPI00646687 19 POF1B Isoform 2 of 14 2 12 0 Protein POF1B IPI00023728 8 GGH Gamma-glutamyl 14 1 12 0 hydrolase precursor IPI00306301 13 PDHA1 Mitochondrial 13 3 12 1 PDHA1 IPI00006379 12 NOP5/NOP58 Nucleolar 13 0 12 0 protein 5 IPI00171692 7 ABHD11 Isoform 1 of 13 2 12 0 Abhydrolase domain- containing protein 11 IPI00329719 25 MYO1D Isoform 1 of 12 4 12 0 Myosin-Id IPI00216225 16 ITGA6 Isoform Alpha- 12 2 12 0 6X1X2A of Integrin alpha-6 precursor IPI00103994 15 LARS Leucyl-tRNA 12 2 12 0 synthetase, cytoplasmic IPI00005198 8 ILF2 Interleukin 12 1 12 0 enhancer-binding factor 2 IPI00783625 5 SERPINB5 Serpin B5 12 0 12 0 precursor IPI00789324 22 JUP JUP protein 11 2 12 0 IPI00871852 21 EIF4A1 46 kDa protein 11 2 12 0 IPI00646493 28 COPA coatomer protein 11 2 12 0 complex, subunit alpha isoform 1 IPI00783982 20 COPG Coatomer 11 2 12 0 subunit gamma IPI00295851 23 COPB1 Coatomer 11 2 12 0 subunit beta IPI00007928 17 PRPF8 Pre-mRNA- 11 1 12 0 processing-splicing factor 8 IPI00008164 17 PREP Prolyl 11 1 12 0 endopeptidase IPI00848161 15 BAT1 Isoform 1 of 10 1 12 0 Spliceosome RNA helicase BAT1 IPI00844578 26 DHX9 ATP-dependent 10 2 12 0 RNA helicase A IPI00383680 19 RPN2 Ribophorin II 10 2 12 0 IPI00011253 14 RPS3 40S ribosomal 10 1 12 0 protein S3 IPI00300371 13 SF3B3 Isoform 1 of 10 0 12 0 Splicing factor 3B subunit 3 IPI00893013 11 XPO1 123 kDa protein 10 1 12 0 IPI00790342 11 RPL6 60S ribosomal 10 2 12 0 protein L6 IPI00011511 7 CECR5 Isoform 2 of Cat 10 4 12 0 eye syndrome critical region protein 5 precursor IPI00023542 4 TMED9 transmembrane 10 0 12 0 emp24 protein transport domain containing 9 IPI00073772 8 FBP1 Fructose-1,6- 9 4 12 0 bisphosphatase 1 IPI00893057 16 PDXDC1 87 kDa 8 3 12 0 protein IPI00029012 15 EIF3A Eukaryotic 8 1 12 0 translation initiation factor 3 subunit A IPI00003833 10 MTCH2 Mitochondrial 8 2 12 0 carrier homolog 2 IPI00792875 3 SERPINB5 14 kDa 8 2 12 0 protein IPI00306960 9 NARS Asparaginyl- 7 1 12 0 tRNA synthetase, cytoplasmic IPI00012912 12 CPT2 Carnitine O- 6 5 12 0 palmitoyltransferase 2, mitochondrial precursor IPI00219518 4 ARL1 ADP-ribosylation 6 1 12 0 factor-like protein 1 IPI00236556 23 MPO Isoform H7 of 24 2 11 0 Myeloperoxidase precursor IPI00218993 20 HSPH1 Isoform Beta of 21 3 11 0 Heat shock protein 105 kDa IPI00477179 8 DDX21 Isoform 2 of 17 1 11 0 Nucleolar RNA helicase 2 IPI00215801 10 RBM39 Isoform 2 of 15 1 11 0 RNA-binding protein 39 IPI00554788 32 KRT18 Keratin, type I 13 2 11 0 cytoskeletal 18 IPI00893918 14 VARS Valyl-tRNA 13 2 11 0 synthetase IPI00877938 11 IARS isoleucyl-tRNA 13 1 11 0 synthetase IPI00641181 2 MARCKSL1 13 0 11 0 MARCKS-related protein IPI00401990 17 ACSL5 acyl-CoA 12 2 11 0 synthetase long-chain family member 5 isoform a IPI00396435 16 DHX15 Putative pre- 12 2 11 0 mRNA-splicing factor ATP-dependent RNA helicase DHX15 IPI00026089 14 SF3B1 Splicing factor 12 1 11 0 3B subunit 1 IPI00005158 14 LONP1 Lon protease 12 2 11 0 homolog, mitochondrial precursor IPI00479262 12 EIF4G1 Isoform B of 12 1 11 0 Eukaryotic translation initiation factor 4 gamma 1 IPI00644431 12 DDX39 ATP-dependent 11 2 11 0 RNA helicase DDX39 IPI00028931 26 DSG2 Desmoglein-2 11 4 11 0 precursor IPI00013452 20 EPRS Bifunctional 11 3 11 0 aminoacyl-tRNA synthetase IPI00418313 17 ILF3 Isoform 4 of 11 3 11 0 Interleukin enhancer- binding factor 3 IPI00017376 7 SEC23B Protein 11 2 11 0 transport protein Sec23B IPI00297084 8 DDOST dolichyl- 11 1 11 0 diphosphooligosaccharide- protein glycosyltransferase precursor IPI00411937 5 NOL5A Nucleolar 11 1 11 1 protein 5A IPI00221091 5 RPS15A 40S ribosomal 11 0 11 0 protein S15a IPI00015872 3 TSPAN8 Tetraspanin-8 11 7 11 0 IPI00784044 11 MCCC2 Isoform 1 of 10 3 11 0 Methylcrotonoyl-CoA carboxylase beta chain, mitochondrial precursor IPI00013485 10 RPS2 40S ribosomal 10 2 11 0 protein S2 IPI00006684 7 API5 58 kDa protein 10 2 11 0 IPI00028004 5 PSMB3 Proteasome 10 2 11 0 subunit beta type-3 IPI00019385 5 SSR4 Translocon- 10 2 11 0 associated protein subunit delta precursor IPI00016608 5 TMED2 10 1 11 0 Transmembrane emp24 domain-containing protein 2 precursor IPI00329791 5 DDX46 cDNA 10 0 11 0 FLJ78679, highly similar to Homo sapiens DEAD (Asp-Glu-Ala- Asp) box polypeptide 46 (DDX46), mRNA IPI00215948 35 CTNNA1 Isoform 1 of 9 3 11 0 Catenin alpha-1 IPI00217952 29 GFPT1 Isoform 1 of 9 2 11 0 Glucosamine--fructose- 6-phosphate aminotransferase [isomerizing] 1 IPI00220834 18 XRCC5 ATP-dependent 9 1 11 0 DNA helicase 2 subunit 2 IPI00514622 3 RANBP6 Ran-binding 9 0 11 0 protein 6 IPI00027252 16 PHB2 Prohibitin-2 9 2 11 0 IPI00020672 13 DPP3; BBS1 Isoform 1 9 3 11 0 of Dipeptidyl-peptidase 3 IPI00017895 13 GPD2 Isoform 1 of 9 2 11 0 Glycerol-3-phosphate dehydrogenase, mitochondrial precursor IPI00872756 10 ASL 58 kDa protein 9 4 11 0 IPI00329598 8 HSD17B11 Estradiol 9 3 11 1 17-beta-dehydrogenase 11 precursor IPI00013296 6 RPS18; LOC100130553 9 1 11 0 40S ribosomal protein S18 IPI00477831 14 ERAP1 Isoform 1 of 8 2 11 0 Endoplasmic reticulum aminopeptidase 1 IPI00303207 7 ABCE1 ATP-binding 8 0 11 0 cassette sub-family E member 1 IPI00847192 6 RPS9 protein 8 1 11 0 (Fragment) IPI00441344 5 GLB1 Isoform 1 of 8 1 11 0 Beta-galactosidase precursor IPI00418497 3 TIMM50 Isoform 2 of 8 0 11 0 Import inner membrane translocase subunit TIM50, mitochondrial precursor IPI00646182 40 ATP1A1 ATPase, 7 3 11 0 Na+/K+ transporting, alpha 1 polypeptide IPI00009634 20 SQRDL Sulfide:quinone 7 3 11 0 oxidoreductase, mitochondrial precursor IPI00304171 12 H2AFY Isoform 2 of 7 1 11 0 Core histone macro- H2A.1 IPI00022887 6 ERGIC1 Isoform 1 of 7 5 11 0 Endoplasmic reticulum- Golgi intermediate compartment protein 1 IPI00171626 2 LPCAT1 1- 7 0 11 0 acylglycerophosphocholine O-acyltransferase 1 IPI00332371 20 PFKL Isoform 1 of 6- 6 2 11 0 phosphofructokinase, liver type IPI00018206 15 GOT2 Aspartate 6 3 11 0 aminotransferase, mitochondrial precursor IPI00643591 5 AP1G1 AP-1 complex 6 1 11 0 subunit gamma-1 IPI00413860 2 STRBP Isoform 2 of 6 0 11 0 Spermatid perinuclear RNA-binding protein IPI00014625 42 CLCA1 Calcium- 4 13 11 1 activated chloride channel regulator 1 precursor IPI00644231 14 CYFIP1 Isoform 1 of 4 2 11 0 Cytoplasmic FMR1- interacting protein 1 IPI00470631 4 COQ9 Isoform 1 of 4 3 11 0 Ubiquinone biosynthesis protein COQ9, mitochondrial precursor IPI00003968 9 NDUFA9 NADH 3 4 11 0 dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9, mitochondrial precursor IPI00221234 14 ALDH7A1 Similar to 2 5 11 0 Antiquitin IPI00007047 6 S100A8 Protein S100- 29 0 10 0 A8 IPI00027462 6 S100A9 Protein S100- 27 1 10 0 A9 IPI00021700 8 PCNA Proliferating cell 18 0 10 0 nuclear antigen IPI00297579 7 CBX3; LOC653972 18 1 10 0 Chromobox protein homolog 3 IPI00642046 4 RSL1D1 RSL1D1 18 1 10 0 protein IPI00002520 14 SHMT2 Serine 17 2 10 0 hydroxymethyltransferase, mitochondrial precursor IPI00219871 2 LSM8 U6 snRNA- 16 2 10 1 associated Sm-like protein LSm8 IPI00030275 17 TRAP1 Heat shock 15 2 10 0 protein 75 kDa, mitochondrial precursor IPI00893035 8 CAD Putative 15 2 10 0 uncharacterized protein CAD IPI00553131 9 GALE UDP-glucose 4- 14 2 10 0 epimerase IPI00217477 3 HMGB3 High mobility 14 3 10 0 group protein B3 IPI00295992 9 ATAD3A Isoform 2 of 13 2 10 0 ATPase family AAA domain-containing protein 3A IPI00646721 9 USP7 Ubiquitin 13 9 10 0 carboxyl-terminal hydrolase IPI00465044 8 RCC2 Protein RCC2 13 1 10 0 IPI00420014 17 ASCC3L1 Isoform 1 of 12 0 10 0 U5 small nuclear ribonucleoprotein 200 kDa helicase IPI00027444 16 SERPINB1 Leukocyte 12 2 10 0 elastase inhibitor IPI00216308 14 VDAC1 Voltage- 12 2 10 0 dependent anion- selective channel protein 1 IPI00334175 14 PTBP1 Isoform 2 of 12 2 10 0 Polypyrimidine tract- binding protein 1 IPI00218466 8 SEC61A1 Isoform 1 of 12 1 10 0 Protein transport protein Sec61 subunit alpha isoform 1 IPI00019912 24 HSD17B4 Peroxisomal 11 1 10 0 multifunctional enzyme type 2 IPI00002557 4 COPG2 Coatomer 11 3 10 0 subunit gamma-2 IPI00449049 16 PARP1 Poly [ADP- 11 1 10 0 ribose] polymerase 1 IPI00014238 10 KARS Lysyl-tRNA 11 2 10 0 synthetase IPI00008530 10 RPLP0 60S acidic 11 2 10 0 ribosomal protein P0 IPI00009950 8 LMAN2 Vesicular 11 2 10 0 integral-membrane protein VIP36 precursor IPI00026202 6 RPL18A 60S ribosomal 11 2 10 0 protein L18a IPI00018597 3 SYK Isoform Long of 11 0 10 0 Tyrosine-protein kinase SYK IPI00216951 21 DARS Aspartyl-tRNA 10 2 10 0 synthetase, cytoplasmic IPI00747497 14 EEF1G 50 kDa protein 10 2 10 0 IPI00032038 14 CPT1A Isoform 1 of 10 7 10 0 Carnitine O- palmitoyltransferase I, liver isoform IPI00297492 10 STT3A Dolichyl- 10 1 10 0 diphosphooligosaccharide-- protein glycosyltransferase subunit STT3A IPI00549672 8 PSMD13 HSPC027 10 0 10 0 IPI00221089 7 RPS13 40S ribosomal 10 1 10 0 protein S13 IPI00152377 7 STT3B Dolichyl- 10 0 10 0 diphosphooligosaccharide-- protein glycosyltransferase subunit STT3B IPI00028055 6 TMED10 10 2 10 0 Transmembrane emp24 domain-containing protein 10 precursor IPI00032139 3 SERPINB9 Serpin B9 10 1 10 0 IPI00290089 28 CDH17 Cadherin-17 9 5 10 0 precursor IPI00797038 21 PCK2 mitochondrial 9 2 10 0 phosphoenolpyruvate carboxykinase 2 isoform 1 precursor IPI00004860 18 RARS Isoform 9 2 10 0 Complexed of Arginyl- tRNA synthetase, cytoplasmic IPI00026665 13 QARS Glutaminyl- 9 1 10 0 tRNA synthetase IPI00017283 10 IARS2 Isoleucyl-tRNA 9 2 10 0 synthetase, mitochondrial precursor IPI00030847 7 TM9SF3 9 1 10 0 Transmembrane 9 superfamily member 3 precursor IPI00014361 5 TSTA3 GDP-L-fucose 9 0 10 0 synthetase IPI00008298 1 DEFA5 Defensin-5 9 0 10 0 precursor IPI00033022 15 DNM2 Isoform 1 of 8 1 10 0 Dynamin-2 IPI00220847 19 ITGB4 Isoform Beta-4D 8 5 10 0 of Integrin beta-4 precursor IPI00847318 15 PKP2 plakophilin 2 8 5 10 0 isoform 2a IPI00215911 11 APEX1 DNA-(apurinic 8 1 10 0 or apyrimidinic site) lyase IPI00874185 10 HIBCH 46 kDa protein 8 5 10 0 IPI00182533 6 RPL28 60S ribosomal 8 6 10 0 protein L28 IPI00296909 6 PARP4 Poly [ADP- 8 2 10 0 ribose] polymerase 4 IPI00023876 5 CASP6 Isoform Alpha 8 3 10 0 of Caspase-6 precursor IPI00744194 5 Similar to 7 4 10 0 Sodium/potassium- transporting ATPase alpha-1 chain precursor IPI00302925 28 CCT8 59 kDa protein 7 2 10 0 IPI00100160 19 CAND1 Isoform 1 of 7 1 10 0 Cullin-associated NEDD8-dissociated protein 1 IPI00337494 13 SLC25A24 Isoform 1 of 7 3 10 0 Calcium-binding mitochondrial carrier protein SCaMC-1 IPI00747849 6 ATP1B1 Isoform 1 of 7 3 10 0 Sodium/potassium- transporting ATPase subunit beta-1 IPI00008433 5 RPS5 40S ribosomal 7 2 10 0 protein S5 IPI00008475 3 HMGCS1 6 4 10 0 Hydroxymethylglutaryl- CoA synthase, cytoplasmic IPI00337541 18 NNT NAD(P) 6 2 10 0 transhydrogenase, mitochondrial precursor IPI00876999 12 PRDX5 Uncharacterized 6 2 10 0 protein PRDX5 (Fragment) IPI00007247 12 PCCB Propionyl-CoA 6 5 10 0 carboxylase beta chain, mitochondrial precursor IPI00032851 5 COPZ1 Coatomer 6 4 10 0 subunit zeta-1 IPI00845474 5 BAX BCL2-associated 6 2 10 0 X protein isoform sigma IPI00152981 3 ACAD9 Acyl-CoA 6 0 10 0 dehydrogenase family member 9, mitochondrial precursor IPI00172656 1 UBXD8 UBX domain- 6 1 10 0 containing protein 8 IPI00008934 20 HMGCS2 5 12 10 0 Hydroxymethylglutaryl- CoA synthase, mitochondrial precursor IPI00790115 11 SLC25A3 cDNA 5 1 10 0 FLJ90278 fis, clone NT2RP1000325, highly similar to Phosphate carrier protein, mitochondrialprecursor IPI00219729 9 SLC25A11 5 4 10 0 Mitochondrial 2- oxoglutarate/malate carrier protein IPI00025277 5 PDCD6 Programmed 5 0 10 0 cell death protein 6 IPI00554701 2 UCRC Cytochrome b-c1 4 0 10 0 complex subunit 9 IPI00008301 2 DEFA6 Defensin-6 4 0 10 0 precursor IPI00456969 88 DYNC1H1 Cytoplasmic 3 1 10 0 dynein 1 heavy chain 1 IPI00220663 17 HK1 Isoform 2 of 3 1 10 0 Hexokinase-1 IPI00719600 7 CYFIP2 Isoform 2 of 3 2 10 0 Cytoplasmic FMR1- interacting protein 2 IPI00398758 5 DCI Isoform 2 of 3,2- 3 5 10 0 trans-enoyl-CoA isomerase, mitochondrial precursor IPI00020510 3 CISD1 CDGSH iron 1 1 10 0 sulfur domain- containing protein 1 IPI00294443 2 CLIC5 Isoform 1 of 21 0 9 0 Chloride intracellular channel protein 5 IPI00031564 7 C7orf24 17 1 9 0 Uncharacterized protein C7orf24 IPI00871140 10 NP Purine nucleoside 16 1 9 0 phosphorylase IPI00291510 9 IMPDH2 Inosine-5′- 15 1 9 0 monophosphate dehydrogenase 2 IPI00792186 5 ABCF1 ATP-binding 15 0 9 0 cassette, sub-family F (GCN20), member 1 IPI00029744 4 SSBP1 Single-stranded 15 3 9 0 DNA-binding protein, mitochondrial precursor IPI00166680 2 MINK1 Isoform 3 of 15 1 9 0 Misshapen-like kinase 1 IPI00152441 3 HM13 Isoform 1 of 14 0 9 0 Minor histocompatibility antigen H13 IPI00303318 8 FAM49B Protein 13 2 9 0 FAM49B IPI00643166 4 PGM3 Isoform 2 of 13 0 9 0 Phosphoacetylglucosamine mutase IPI00641950 14 GNB2L1 Lung cancer 12 2 9 0 oncogene 7 IPI00293464 11 DDB1 DNA damage- 12 1 9 0 binding protein 1 IPI00783097 10 GARS Glycyl-tRNA 12 2 9 0 synthetase IPI00219617 4 PRPS2 Isoform 1 of 12 1 9 0 Ribose-phosphate pyrophosphokinase 2 IPI00018415 7 TM9SF2 12 1 9 0 Transmembrane 9 superfamily member 2 precursor IPI00550032 4 LOC653232; RPL15 12 0 9 0 Ribosomal protein L15 pseudogene 3 IPI00013933 65 DSP Isoform DPI of 11 2 9 0 Desmoplakin IPI00009032 15 SSB Lupus La protein 11 1 9 0 IPI00792100 10 C14orf166 CLE 11 1 9 0 IPI00010491 3 RAB27B Ras-related 11 2 9 0 protein Rab-27B IPI00302850 3 SNRPD1 Small nuclear 11 1 9 0 ribonucleoprotein Sm D1 IPI00644712 20 XRCC6 ATP-dependent 10 1 9 0 DNA helicase 2 subunit 1 IPI00025874 20 RPN1 Dolichyl- 10 2 9 0 diphosphooligosaccharide-- protein glycosyltransferase 67 kDa subunit precursor IPI00744889 9 CDH1 E-cadherin 10 2 9 0 IPI00002372 8 ABCD3 Isoform 1 of 10 4 9 0 ATP-binding cassette sub-family D member 3 IPI00289601 4 HDAC2 histone 10 2 9 0 deacetylase 2 IPI00328867 5 SRC Isoform 2 of Proto- 10 0 9 0 oncogene tyrosine- protein kinase Src IPI00872474 3 LYN LYN protein 10 0 9 0 (Fragment) IPI00031804 8 VDAC3 Isoform 1 of 10 2 9 0 Voltage-dependent anion-selective channel protein 3 IPI00654777 7 EIF3F Eukaryotic 10 0 9 0 translation initiation factor 3 subunit 5 IPI00440703 6 GSTK1 GSTK1 protein 10 3 9 0 IPI00885106 3 TM9SF4 Isoform 2 of 10 0 9 0 Transmembrane 9 superfamily member 4 precursor IPI00029267 2 SNRPB2 U2 small 10 2 9 0 nuclear ribonucleoprotein B″ IPI00414717 12 GLG1 golgi apparatus 9 1 9 0 protein 1 IPI00216293 11 TST Thiosulfate 9 6 9 0 sulfurtransferase IPI00029629 11 TRIM25 Tripartite 9 2 9 0 motif-containing protein 25 IPI00219147 4 CSDA Isoform 2 of 9 1 9 0 DNA-binding protein A IPI00219953 9 CMPK1 cytidine 9 3 9 0 monophosphate (UMP- CMP) kinase 1, cytosolic IPI00827508 7 RPL10A 25 kDa protein 9 2 9 0 IPI00014053 6 TOMM40 Isoform 1 of 9 0 9 0 Mitochondrial import receptor subunit TOM40 homolog IPI00299048 20 IQGAP2 Isoform 1 of 8 2 9 0 Ras GTPase-activating- like protein IQGAP2 IPI00242956 70 FCGBP IgGFc-binding 8 9 9 2 protein precursor IPI00291467 18 SLC25A6 ADP/ATP 8 2 9 0 translocase 3 IPI00290566 22 TCP1 T-complex 8 2 9 0 protein 1 subunit alpha IPI00470502 14 PPA2 Isoform 2 of 8 1 9 0 Inorganic pyrophosphatase 2, mitochondrial precursor IPI00003482 12 DECR1 2,4-dienoyl- 8 4 9 0 CoA reductase, mitochondrial precursor IPI00456750 10 FAM129B Niban-like 8 3 9 0 protein 1 IPI00303158 10 CMAS Isoform 1 of N- 8 2 9 0 acylneuraminate cytidylyltransferase IPI00001466 8 EML4 Echinoderm 8 2 9 0 microtubule-associated protein-like 4 IPI00105598 7 PSMD11 Proteasome 8 1 9 0 26S non-ATPase subunit 11 variant (Fragment) IPI00011916 4 JTV1 Multisynthetase 8 1 9 0 complex auxiliary component p38 IPI00100460 3 DARS2 Aspartyl-tRNA 8 1 9 0 synthetase, mitochondrial precursor IPI00030706 3 AHSA1 Activator of 90 kDa 8 1 9 0 heat shock protein ATPase homolog 1 IPI00874145 3 DKC1 Uncharacterized 8 0 9 0 protein DKC1 (Fragment) IPI00177817 24 ATP2A2 Isoform 7 2 9 0 SERCA2A of Sarcoplasmic/endoplasmic reticulum calcium ATPase 2 IPI00409717 13 EIF4A2 Isoform 2 of 7 1 9 0 Eukaryotic initiation factor 4A-II IPI00719752 15 EIF3B Isoform 2 of 7 2 9 0 Eukaryotic translation initiation factor 3 subunit B IPI00001091 12 AFG3L2 AFG3-like 7 7 9 0 protein 2 IPI00026530 10 LMAN1 Protein 7 2 9 0 ERGIC-53 precursor IPI00007676 9 HSD17B12 Estradiol 7 2 9 0 17-beta-dehydrogenase 12 IPI00291930 8 CLINT1 Isoform 1 of 7 2 9 0 Clathrin interactor 1 IPI00293853 7 GPA33 Cell surface 7 6 9 0 A33 antigen precursor IPI00797738 5 COX6B1 12 kDa 7 2 9 0 protein IPI00885058 3 MBOAT7 Isoform 2 of 7 5 9 0 Membrane-bound O- acyltransferase domain- containing protein 7 IPI00455383 68 CLTC Isoform 2 of 6 2 9 0 Clathrin heavy chain 1 IPI00102864 12 HK2 Hexokinase-2 6 2 9 0 IPI00018931 16 VPS35 Vacuolar protein 6 0 9 1 sorting-associated protein 35 IPI00010157 8 MAT2A S- 6 1 9 1 adenosylmethionine synthetase isoform type-2 IPI00328715 6 MTDH Protein LYRIC 6 1 9 0 IPI00171573 5 CCDC109A Isoform 1 6 0 9 0 of Coiled-coil domain- containing protein 109A IPI00411426 5 VPS26A Vacuolar 6 1 9 0 protein sorting- associated protein 26A IPI00017767 2 MGST2 Microsomal 6 2 9 0 glutathione S-transferase 2 IPI00556311 2 DUOX2 Dual oxidase 2 6 0 9 0 variant (Fragment) IPI00011201 10 ME2 NAD-dependent 5 3 9 0 malic enzyme, mitochondrial precursor IPI00329672 5 MYO1E Myosin-Ie 5 1 9 0 IPI00020928 4 TFAM Transcription 5 4 9 0 factor A, mitochondrial precursor IPI00009104 12 RUVBL2 RuvB-like 2 4 0 9 0 IPI00744115 12 PCCA propionyl- 4 3 9 0 Coenzyme A carboxylase, alpha polypeptide precursor IPI00219029 10 GOT1 Aspartate 4 3 9 0 aminotransferase, cytoplasmic IPI00029264 7 CYC1 Cytochrome c1, 4 2 9 0 heme protein, mitochondrial precursor IPI00027448 5 ATP5L ATP synthase 4 2 9 0 subunit g, mitochondrial IPI00215920 2 ARF6 ADP-ribosylation 4 0 9 0 factor 6 IPI00220244 1 TRIM23 Isoform Beta 4 0 9 0 of GTP-binding protein ARD-1 IPI00006674 7 ABCC3 Isoform 3 of 3 4 9 0 Canalicular multispecific organic anion transporter 2 IPI00843876 7 TNPO1 Transportin-1 3 0 9 0 IPI00023001 3 C3orf28 E2-induced 3 5 9 0 gene 5 protein IPI00019038 4 LYZ Lysozyme C 17 2 8 0 precursor IPI00022462 14 TFRC Transferrin 16 0 8 0 receptor protein 1 IPI00554722 7 LOC442497; SLC3A2 16 1 8 0 solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 isoform e IPI00010341 6 PRG2 Bone marrow 16 7 8 0 proteoglycan precursor IPI00010320 2 CBX1 Chromobox 16 1 8 0 protein homolog 1 IPI00006690 27 EPX Eosinophil 15 8 8 0 peroxidase precursor IPI00099110 4 DMBT1 Isoform 1 of 15 0 8 1 Deleted in malignant brain tumors 1 protein precursor IPI00008240 8 MARS Methionyl-tRNA 14 1 8 0 synthetase, cytoplasmic IPI00414676 41 HSP90AB1 Heat shock 13 2 8 0 protein HSP 90-beta IPI00140420 27 SND1 Staphylococcal 13 2 8 0 nuclease domain- containing protein 1 IPI00807557 14 PA2G4 PA2G4 protein 13 2 8 0 (Fragment) IPI00215879 6 SFRS6 Isoform SRP55- 13 1 8 0 3 of Splicing factor, arginine/serine-rich 6 IPI00301936 8 ELAVL1 ELAV-like 13 1 8 0 protein 1 IPI00025039 7 FBL rRNA 2′-O- 13 2 8 0 methyltransferase fibrillarin IPI00025427 4 RNASE3 Eosinophil 13 6 8 1 cationic protein precursor IPI00873179 4 CLC Uncharacterized 13 6 8 0 protein CLC (Fragment) IPI00655650 3 LOC728937; RPS26 40S 13 1 8 0 ribosomal protein S26 IPI00888987 2 LOC345630 similar to 13 1 8 0 hCG1641252 IPI00026952 13 PKP3 Plakophilin-3 12 1 8 0 IPI00009328 12 EIF4A3 Eukaryotic 12 2 8 0 initiation factor 4A-III IPI00759824 6 ANP32B Isoform 2 of 12 2 8 1 Acidic leucine-rich nuclear phosphoprotein 32 family member B IPI00029048 9 TTLL12 Tubulin-- 12 3 8 0 tyrosine ligase-like protein 12 IPI00029764 6 SF3A3 Splicing factor 12 1 8 0 3A subunit 3 IPI00303954 4 CYB5B cytochrome b5 12 2 8 0 outer mitochondrial membrane precursor IPI00007402 4 IPO7 Importin-7 12 0 8 0 IPI00012382 2 SNRPA U1 small 12 2 8 0 nuclear ribonucleoprotein A IPI00446377 2 ENG cDNA FLJ41744 12 0 8 0 fis, clone HSYRA2005496, highly similar to ENDOGLIN IPI00186290 45 EEF2 Elongation factor 2 11 2 8 0 IPI00304596 18 NONO Non-POU 11 2 8 0 domain-containing octamer-binding protein IPI00220219 17 COPB2 Coatomer 11 2 8 0 subunit beta′ IPI00003918 16 RPL4 60S ribosomal 11 2 8 0 protein L4 IPI00020632 14 ASS1 Argininosuccinate 11 3 8 0 synthase IPI00396661 5 CYP2S1 Isoform 1 of 11 0 8 0 Cytochrome P450 2S1 IPI00005589 5 hCG_18290 11 1 8 0 Uncharacterized protein ENSP00000275524 IPI00178440 4 EEF1B2 Elongation 11 1 8 0 factor 1-beta IPI00894416 4 BZW2 47 kDa protein 11 0 8 0 IPI00793862 3 SHMT1 Serine 11 0 8 0 hydroxymethyltransferase IPI00029750 3 RPS24 Isoform 1 of 40S 11 1 8 0 ribosomal protein S24 IPI00873680 3 EIF4E Uncharacterized 11 2 8 0 protein EIF4E (Fragment) IPI00784154 45 HSPD1 60 kDa heat 10 3 8 0 shock protein, mitochondrial precursor IPI00219005 15 FKBP4 FK506-binding 10 2 8 0 protein 4 IPI00003519 13 EFTUD2 116 kDa U5 10 1 8 0 small nuclear ribonucleoprotein component IPI00013774 4 HDAC1 Histone 10 3 8 0 deacetylase 1 IPI00428967 6 TICAM2; TMED7 Toll- 10 1 8 0 like receptor adapter molecule 2 IPI00219445 5 PSME3 Isoform 2 of 10 1 8 0 Proteasome activator complex subunit 3 IPI00029731 4 RPL35A 60S ribosomal 10 2 8 0 protein L35a IPI00025329 3 RPL19 60S ribosomal 10 4 8 0 protein L19 IPI00455757 3 Similar to 60S 10 2 8 0 ribosomal protein L35 IPI00009407 2 DAD1 Dolichyl- 10 0 8 0 diphosphooligosaccharide-- protein glycosyltransferase subunit DAD1 IPI00010951 33 EPPK1 Epiplakin 9 1 8 0 IPI00658109 16 LOC100133623; CKMT1B; 9 9 8 0 CKMT1A Creatine kinase, ubiquitous mitochondrial precursor IPI00293721 9 AKR7A3 Aflatoxin B1 9 4 8 0 aldehyde reductase member 3 IPI00642211 15 RNPEP Aminopeptidase B 9 2 8 0 IPI00299608 14 PSMD1 Isoform 1 of 9 2 8 0 26S proteasome non- ATPase regulatory subunit 1 IPI00017726 13 HSD17B10 Isoform 1 of 9 2 8 0 3-hydroxyacyl-CoA dehydrogenase type-2 IPI00479722 13 PSME1 Proteasome 9 1 8 0 activator complex subunit 1 IPI00009750 12 LGALS4 Galectin-4 9 6 8 0 IPI00334190 10 STOML2 Stomatin-like 9 2 8 0 protein 2 IPI00853220 10 SEC31A Isoform 6 of 9 2 8 0 Protein transport protein Sec31A IPI00030131 9 TMPO Isoform Beta of 9 2 8 0 Lamina-associated polypeptide 2, isoforms beta/gamma IPI00640155 8 PSMB8 proteasome beta 9 3 8 0 8 subunit isoform E2 proprotein IPI00221222 7 SUB1 Activated RNA 9 2 8 0 polymerase II transcriptional coactivator p15 IPI00465361 6 RPL13 60S ribosomal 9 2 8 0 protein L13 IPI00005537 6 MRPL12 39S ribosomal 9 4 8 1 protein L12, mitochondrial precursor IPI00293564 5 HMGCL 9 3 8 0 Hydroxymethylglutaryl- CoA lyase, mitochondrial precursor IPI00785096 4 BZW1 Isoform 1 of 9 1 8 0 Basic leucine zipper and W2 domain-containing protein 1 IPI00100656 4 GPSN2 Isoform 1 of 9 0 8 1 Synaptic glycoprotein SC2 IPI00009922 4 C14orf156 SRA stem- 9 1 8 0 loop-interacting RNA- binding protein, mitochondrial precursor IPI00007188 17 SLC25A5 ADP/ATP 8 2 8 0 translocase 2 IPI00017375 9 SEC23A Protein 8 1 8 0 transport protein Sec23A IPI00873762 3 TAF15 65 kDa protein 8 0 8 0 IPI00885213 7 TXNRD1 Isoform 6 of 8 1 8 3 Thioredoxin reductase 1, cytoplasmic IPI00329352 6 NOMO1; NOMO3 8 3 8 0 Nodal modulator 1 precursor IPI00029628 3 RCN2 Reticulocalbin-2 8 0 8 0 precursor IPI00797230 2 RPL8 32 kDa protein 8 3 8 0 IPI00220835 2 SEC61B Protein 8 2 8 0 transport protein Sec61 subunit beta IPI00784366 13 AP2B1 Isoform 2 of 7 0 8 0 AP-2 complex subunit beta-1 IPI00220994 5 H2AFY2 Core histone 7 1 8 0 macro-H2A.2 IPI00012268 12 PSMD2 26S proteasome 7 2 8 0 non-ATPase regulatory subunit 2 IPI00013068 12 EIF3E Eukaryotic 7 0 8 0 translation initiation factor 3 subunit E IPI00472054 11 FAM120A Isoform A of 7 4 8 0 Constitutive coactivator of PPAR-gamma-like protein 1 IPI00383046 6 CMBL 7 8 8 1 Carboxymethylenebutenolidase homolog IPI00031820 5 FARSA Phenylalanyl- 7 2 8 0 tRNA synthetase alpha chain IPI00004845 4 NIPSNAP3A Protein 7 3 8 0 NipSnap homolog 3A IPI00018465 24 CCT7 T-complex 6 2 8 0 protein 1 subunit eta IPI00027442 19 AARS Alanyl-tRNA 6 3 8 0 synthetase, cytoplasmic IPI00017592 9 LETM1 Leucine zipper- 6 2 8 0 EF-hand-containing transmembrane protein 1, mitochondrial precursor IPI00300050 8 HSD11B2 6 13 8 2 Corticosteroid 11-beta- dehydrogenase isozyme 2 IPI00306516 8 TIMM44 Import inner 6 1 8 0 membrane translocase subunit TIM44, mitochondrial precursor IPI00293267 5 LGALS9 Isoform Short 6 3 8 0 of Galectin-9 IPI00021258 7 ARFIP1 Isoform B of 6 2 8 1 Arfaptin-1 IPI00303568 5 PTGES2 Prostaglandin 6 1 8 0 E synthase 2 IPI00003870 5 CLPP Putative ATP- 6 2 8 0 dependent Clp protease proteolytic subunit, mitochondrial precursor IPI00843910 4 FUCA1 Tissue alpha-L- 6 16 8 4 fucosidase precursor IPI00452747 4 LOC653566 Similar to 6 1 8 0 Signal peptidase complex subunit 2 IPI00304612 2 RPL13A 60S ribosomal 6 1 8 0 protein L13a IPI00871366 9 RAB1B Small GTP- 5 1 8 0 binding protein IPI00006451 9 NSF Vesicle-fusing 5 0 8 1 ATPase IPI00025239 6 NDUFS2 NADH 5 2 8 0 dehydrogenase [ubiquinone] iron-sulfur protein 2, mitochondrial precursor IPI00550644 5 LL22NC03-5H6.5 5 3 8 0 UPF0530 protein IPI00016077 3 GBAS Protein NipSnap 5 6 8 0 homolog 2 IPI00031534 2 ST6GALNAC1 Alpha- 5 5 8 0 N-acetylgalactosaminide alpha-2,6- sialyltransferase 1 IPI00025796 9 NDUFS3 NADH 4 1 8 0 dehydrogenase [ubiquinone] iron-sulfur protein 3, mitochondrial precursor IPI00794899 3 37 kDa protein 4 4 8 0 IPI00029054 4 NT5C2 Cytosolic purine 4 0 8 0 5′-nucleotidase IPI00002255 4 LRBA 4 3 8 0 Lipopolysaccharide- responsive and beige- like anchor protein IPI00253050 2 L1TD1 LINE-1 type 4 0 8 0 transposase domain- containing protein 1 IPI00465179 8 PFKM cDNA FLJ44241 3 1 8 0 fis, clone THYMU3008436, highly similar to 6- phosphofructokinase, muscle type IPI00294187 9 PADI2 Protein-arginine 3 13 8 2 deiminase type-2 IPI00102581 7 SULT1B1 3 9 8 0 Sulfotransferase family cytosolic 1B member 1 IPI00023647 4 UBA6 Isoform 1 of 3 2 8 0 Ubiquitin-like modifier- activating enzyme 6 IPI00030320 5 DDX6 Probable ATP- 2 1 8 0 dependent RNA helicase DDX6 IPI00220740 8 NPM1 Isoform 2 of 17 2 7 0 Nucleophosmin IPI00386755 7 ERO1L ERO1-like 17 1 7 1 protein alpha precursor IPI00295542 13 NUCB1 Nucleobindin-1 15 3 7 0 precursor IPI00293655 13 DDX1 ATP-dependent 14 1 7 0 RNA helicase DDX1 IPI00007084 11 SLC25A13 14 2 7 0 Mitochondrial aspartate- glutamate carrier protein IPI00007427 8 AGR2 AGR2 14 5 7 0 IPI00152409 7 AGR3 Anterior gradient 14 4 7 0 protein 3 homolog precursor IPI00004573 27 PIGR Polymeric 13 5 7 1 immunoglobulin receptor precursor IPI00021290 15 ACLY ATP-citrate 13 1 7 0 synthase IPI00456919 14 HUWE1 Isoform 1 of 13 0 7 0 E3 ubiquitin-protein ligase HUWE1 IPI00015018 13 PPA1 Inorganic 13 2 7 0 pyrophosphatase IPI00218493 8 HPRT1 Hypoxanthine- 13 1 7 0 guanine phosphoribosyltransferase IPI00876962 6 INF2 Isoform 2 of 13 2 7 0 Inverted formin-2 IPI00479997 5 STMN1 Stathmin 13 1 7 0 IPI00791426 5 RPL24 13 kDa protein 13 1 7 0 IPI00215719 4 RPL18 60S ribosomal 13 1 7 0 protein L18 IPI00334713 9 HNRNPAB Isoform 3 12 3 7 0 of Heterogeneous nuclear ribonucleoprotein A/B IPI00012442 8 G3BP1 Ras GTPase- 12 0 7 0 activating protein- binding protein 1 IPI00301311 8 SET Isoform 2 of 12 2 7 0 Protein SET IPI00011913 6 HNRNPA0 12 2 7 0 Heterogeneous nuclear ribonucleoprotein A0 IPI00413673 5 BCLAF1 Isoform 4 of 12 3 7 0 Bcl-2-associated transcription factor 1 IPI00059292 4 MAGOHB Protein 12 0 7 1 mago nashi homolog 2 IPI00409635 3 FAM62B Isoform 2 of 12 0 7 0 Extended synaptotagmin-2 IPI00555747 9 PABPC4 Isoform 2 of 11 2 7 0 Polyadenylate-binding protein 4 IPI00604620 25 NCL Isoform 1 of 11 3 7 0 Nucleolin IPI00000877 24 HYOU1 Hypoxia up- 11 2 7 0 regulated protein 1 precursor IPI00141318 24 CKAP4 Isoform 1 of 11 2 7 0 Cytoskeleton-associated protein 4 IPI00217468 10 HIST1H1B Histone 11 2 7 0 H1.5 IPI00797148 15 HNRNPA1 HNRPA1 11 2 7 0 protein IPI00438229 15 TRIM28 Isoform 1 of 11 1 7 0 Transcription intermediary factor 1- beta IPI00031812 6 YBX1 Nuclease- 11 2 7 0 sensitive element- binding protein 1 IPI00221092 8 RPS16 40S ribosomal 11 2 7 0 protein S16 IPI00396321 8 LRRC59 Leucine-rich 11 3 7 0 repeat-containing protein 59 IPI00009659 4 C20orf77 11 1 7 0 Uncharacterized protein C20orf77 IPI00157790 4 KIAA0368 KIAA0368 11 0 7 0 protein IPI00794978 2 MRPL47 MRPL47 11 1 7 0 protein IPI00004524 2 GCA Grancalcin 11 0 7 0 IPI00217437 7 TTBK2 Tau-tubulin 10 5 7 0 kinase IPI00880104 3 KRT74 59 kDa protein 10 3 7 0 IPI00550661 3 KRT13 Isoform 2 of 10 5 7 0 Keratin, type I cytoskeletal 13 IPI00382470 44 HSP90AA1 heat shock 10 1 7 0 protein 90 kDa alpha (cytosolic), class A member 1 isoform 1 IPI00651677 24 DDX17 Isoform 2 of 10 2 7 0 Probable ATP- dependent RNA helicase DDX17 IPI00017617 20 DDX5 Probable ATP- 10 3 7 0 dependent RNA helicase DDX5 IPI00010471 29 LCP1 Plastin-2 10 1 7 1 IPI00007765 34 HSPA9 Stress-70 10 2 7 0 protein, mitochondrial precursor IPI00298520 16 ARCN1 Putative 10 2 7 0 uncharacterized protein DKFZp686M09245 IPI00217030 14 RPS4X 40S ribosomal 10 2 7 0 protein S4, X isoform IPI00000494 13 RPL5 60S ribosomal 10 2 7 0 protein L5 IPI00012585 10 HEXB Beta- 10 3 7 0 hexosaminidase subunit beta precursor IPI00026105 10 SCP2 Isoform SCPx of 10 4 7 0 Non-specific lipid- transfer protein IPI00008455 8 MYO6 Isoform 2 of 10 1 7 0 Myosin-VI IPI00399183 7 APOB48R Isoform 1 of 10 5 7 1 Apolipoprotein B-100 receptor IPI00744364 6 SFRS7 Uncharacterized 10 4 7 0 protein SFRS7 IPI00465132 5 COPE Coatomer subunit 10 1 7 0 epsilon IPI00607584 5 MYBBP1A Isoform 2 of 10 0 7 0 Myb-binding protein 1A IPI00787692 4 LOC650788 similar to 10 2 7 0 40S ribosomal protein S28 IPI00014808 3 PAFAH1B3 Platelet- 10 0 7 0 activating factor acetylhydrolase IB subunit gamma IPI00019449 3 LOC100133484; HLA- 10 9 7 1 DQB1; HLA- DRB1; hCG_1998957; HLA- DRB4; LOC100133811; LOC100133583; HLA- DRB3; HLA- DRB5; HLA- DQB2; HLA- DRB2; ZNF749; LOC100133661; RNASE2 Non- secretory ribonuclease precursor IPI00217975 31 LMNB1 Lamin-B1 9 3 7 1 IPI00455599 12 HSP90AB2P Heat shock 9 2 7 0 protein 90Bb IPI00853059 14 FUBP1 Isoform 2 of Far 9 1 7 0 upstream element- binding protein 1 IPI00218852 30 VIL1 Villin-1 9 3 7 0 IPI00789551 15 MATR3 9 1 7 0 Uncharacterized protein MATR3 IPI00410693 11 SERBP1 Isoform 1 of 9 4 7 0 Plasminogen activator inhibitor 1 RNA-binding protein IPI00646899 10 RPL10 Ribosomal 9 2 7 0 protein L10 IPI00220362 9 HSPE1 10 kDa heat 9 3 7 0 shock protein, mitochondrial IPI00299573 9 RPL7A 60S ribosomal 9 2 7 0 protein L7a IPI00221354 5 FUS Isoform Short of 9 2 7 0 RNA-binding protein FUS IPI00893715 7 TACSTD1 38 kDa 9 4 7 0 protein IPI00872533 6 CD2AP 76 kDa protein 9 1 7 0 IPI00006181 6 EIF3D Eukaryotic 9 3 7 0 translation initiation factor 3 subunit D IPI00009368 5 SFXN1 Sideroflexin-1 9 2 7 1 IPI00219516 4 GUSB Isoform Short of 9 2 7 0 Beta-glucuronidase precursor IPI00398135 4 hCG_21078 9 2 7 0 hypothetical protein LOC389435 IPI00082310 3 KHDRBS1 Isoform 3 of 9 1 7 0 KH domain-containing, RNA-binding, signal transduction-associated protein 1 IPI00328268 2 EIF4G3 EIF4G3 protein 9 0 7 0 IPI00644079 21 HNRNPU 8 2 7 0 heterogeneous nuclear ribonucleoprotein U isoform a IPI00414980 9 MYO1B Isoform 2 of 8 5 7 4 Myosin-Ib IPI00646304 13 PPIB peptidylprolyl 8 3 7 0 isomerase B precursor IPI00017334 13 PHB Prohibitin 8 1 7 0 IPI00744692 13 TALDO1 Transaldolase 8 1 7 0 IPI00329633 12 TARS Threonyl-tRNA 8 1 7 0 synthetase, cytoplasmic IPI00030009 11 PAPSS2 Isoform A of 8 10 7 1 Bifunctional 3′- phosphoadenosine 5′- phosphosulfate synthetase 2 IPI00027851 7 HEXA Beta- 8 3 7 0 hexosaminidase subunit alpha precursor IPI00013174 7 RBM14 Isoform 1 of 8 3 7 0 RNA-binding protein 14 IPI00024933 7 RPL12 60S ribosomal 8 2 7 0 protein L12 IPI00793375 6 XPNPEP1 Xaa-Pro 8 1 7 0 aminopeptidase 1 IPI00219160 5 RPL34 60S ribosomal 8 4 7 0 protein L34 IPI00010949 5 SIAE Isoform 1 of 8 7 7 1 Sialate O-acetylesterase precursor IPI00005737 4 SURF4 Isoform 1 of 8 1 7 0 Surfeit locus protein 4 IPI00029631 3 ERH Enhancer of 8 0 7 0 rudimentary homolog IPI00029601 15 CTTN Src substrate 7 3 7 0 cortactin IPI00030179 11 RPL7 60S ribosomal 7 3 7 0 protein L7 IPI00644570 3 18 kDa protein 7 4 7 0 IPI00456887 13 HNRNPUL2 7 0 7 0 Heterogeneous nuclear ribonucleoprotein U-like protein 2 IPI00031169 11 RAB2A Ras-related 7 0 7 0 protein Rab-2A IPI00016910 11 EIF3CL; EIF3C 7 3 7 0 Eukaryotic translation initiation factor 3 subunit C IPI00031583 11 USO1 Putative 7 4 7 4 uncharacterized protein DKFZp451D234 IPI00165360 9 MPST 3- 7 3 7 0 mercaptopyruvate sulfurtransferase IPI00025019 7 PSMB1 Proteasome 7 2 7 0 subunit beta type-1 precursor IPI00335930 4 DAZAP1 Isoform 2 of 7 1 7 0 DAZ-associated protein 1 IPI00015029 4 PTGES3 Prostaglandin 7 2 7 0 E synthase 3 IPI00029039 4 REG3A Regenerating 7 0 7 0 islet-derived protein 3 alpha precursor IPI00061525 3 GNPNAT1 7 0 7 0 Glucosamine 6- phosphate N- acetyltransferase IPI00026167 3 NHP2L1 NHP2-like 7 0 7 0 protein 1 IPI00000811 3 PSMB6 Proteasome 7 2 7 1 subunit beta type-6 precursor IPI00790799 3 SEC11A 16 kDa protein 7 0 7 0 IPI00413654 3 SFRS5 Isoform SRP40- 7 2 7 0 4 of Splicing factor, arginine/serine-rich 5 IPI00006092 3 PMM2 7 1 7 0 Phosphomannomutase 2 IPI00328840 3 THOC4 THO complex 7 1 7 0 subunit 4 IPI00216237 3 RPL36 60S ribosomal 7 3 7 1 protein L36 IPI00173589 2 LOC284064 similar to 7 3 7 0 ribosomal protein L29 IPI00009342 64 IQGAP1 Ras GTPase- 6 2 7 0 activating-like protein IQGAP1 IPI00413947 10 AP1B1 Isoform B of 6 0 7 0 AP-1 complex subunit beta-1 IPI00398798 5 H2AFV H2A histone 6 4 7 0 family, member V isoform 3 IPI00303882 13 M6PRBP1 Isoform B of 6 2 7 0 Mannose-6-phosphate receptor-binding protein 1 IPI00790743 5 Protein 6 5 7 0 IPI00021800 8 CASP1 Isoform Alpha 6 5 7 0 of Caspase-1 precursor IPI00060181 7 EFHD2 EF-hand 6 0 7 1 domain-containing protein D2 IPI00017510 6 MT-CO2 Cytochrome c 6 6 7 0 oxidase subunit 2 IPI00006443 6 CRYL1 Lambda- 6 6 7 0 crystallin homolog IPI00020956 6 HDGF Hepatoma- 6 2 7 0 derived growth factor IPI00060200 5 GALM Aldose 1- 6 6 7 0 epimerase IPI00185374 5 PSMD12 26S 6 1 7 0 proteasome non-ATPase regulatory subunit 12 IPI00012340 4 SFRS9 Splicing factor, 6 3 7 1 arginine/serine-rich 9 IPI00016405 4 OCIAD1 Isoform 1 of 6 5 7 0 OCIA domain- containing protein 1 IPI00019329 3 DYNLL1 Dynein light 6 4 7 0 chain 1, cytoplasmic IPI00642816 3 SRP9; hCG_1781062 6 2 7 0 Signal recognition particle 9 kDa protein IPI00013968 3 COX7C Cytochrome c 6 2 7 0 oxidase subunit 7C, mitochondrial precursor IPI00026570 2 COX7A2 Cytochrome c 6 4 7 0 oxidase polypeptide VIIa-liver/heart, mitochondrial precursor IPI00005719 10 RAB1A Isoform 1 of 5 2 7 0 Ras-related protein Rab- 1A IPI00374686 6 Uncharacterized protein 5 1 7 0 ENSP00000341227 (Fragment) IPI00219077 15 LTA4H Isoform 1 of 5 2 7 0 Leukotriene A-4 hydrolase IPI00025366 13 CS Citrate synthase, 5 1 7 1 mitochondrial precursor IPI00290110 13 PDCD4 Programmed 5 5 7 0 cell death protein 4 IPI00332828 10 CES2 carboxylesterase 2 5 7 7 5 isoform 1 IPI00182757 7 KIAA1967 Isoform 1 of 5 3 7 0 Protein KIAA1967 IPI00030654 6 CPSF6 Isoform 2 of 5 1 7 0 Cleavage and polyadenylation specificity factor subunit 6 IPI00789848 5 IVD Isovaleryl-CoA 5 7 7 0 dehydrogenase, mitochondrial precursor IPI00024934 4 MUT Methylmalonyl- 5 0 7 0 CoA mutase, mitochondrial precursor IPI00015972 4 COX6C Cytochrome c 5 6 7 0 oxidase polypeptide VIc precursor IPI00031772 3 C15orf48 Normal 5 7 7 0 mucosa of esophagus- specific gene 1 protein IPI00019888 2 ALDH5A1 Succinate- 5 0 7 0 semialdehyde dehydrogenase, mitochondrial precursor IPI00873259 2 ATP5J2 10 kDa protein 5 2 7 0 IPI00005159 11 ACTR2 Actin-related 4 1 7 0 protein 2 IPI00465256 8 AK3 GTP:AMP 4 2 7 1 phosphotransferase mitochondrial IPI00513827 8 ACADM Putative 4 4 7 0 uncharacterized protein DKFZp686M24262 IPI00554811 6 ARPC4; TTLL3 Actin- 4 2 7 0 related protein 2/3 complex subunit 4 IPI00874156 6 OTUB1 Isoform 1 of 4 2 7 0 Ubiquitin thioesterase OTUB1 IPI00024661 4 SEC24C Protein 4 2 7 1 transport protein Sec24C IPI00028387 4 C20orf116 Isoform 1 of 4 1 7 0 Uncharacterized protein C20orf116 precursor IPI00022277 4 CCDC56 Coiled-coil 4 6 7 0 domain-containing protein 56 IPI00024742 2 UQCRQ Cytochrome b- 4 3 7 0 c1 complex subunit 8 IPI00307547 2 C9orf46 4 1 7 0 Uncharacterized protein C9orf46 IPI00029133 10 ATP5F1 ATP synthase 3 3 7 0 subunit b, mitochondrial precursor IPI00651719 6 PBLD MAWD binding 3 2 7 1 protein isoform b IPI00219755 1 SPCS1 Signal peptidase 3 3 7 0 complex subunit 1 IPI00880101 9 CEACAM5 Protein 29 0 6 0 IPI00302944 51 COL12A1 Isoform 4 of 24 3 6 0 Collagen alpha-1(XII) chain precursor IPI00027769 5 ELA2 Leukocyte 23 0 6 0 elastase precursor IPI00026781 46 FASN Fatty acid 20 2 6 0 synthase IPI00304754 5 FERMT1 Isoform 1 of 18 3 6 0 Fermitin family homolog 1 IPI00019472 5 SLC1A5 Neutral amino 16 0 6 0 acid transporter B IPI00026833 5 ADSS Adenylosuccinate 16 0 6 0 synthetase isozyme 2 IPI00026328 3 TXNDC12 Thioredoxin 14 2 6 0 domain-containing protein 12 precursor IPI00009904 36 PDIA4 Protein 13 3 6 0 disulfide-isomerase A4 precursor IPI00216044 8 RALY Isoform 1 of 13 0 6 0 RNA-binding protein Raly IPI00215790 6 RPL38 60S ribosomal 13 2 6 0 protein L38 IPI00872940 6 RPL30 Uncharacterized 13 1 6 0 protein RPL30 (Fragment) IPI00012007 11 AHCY 12 2 6 0 Adenosylhomocysteinase IPI00012069 6 NQO1 NAD 12 1 6 0 IPI00385267 5 SRPR Signal 12 1 6 0 recognition particle receptor subunit alpha IPI00856058 4 RPL31 ribosomal 12 3 6 0 protein L31 isoform 3 IPI00395865 3 RBBP7 Histone-binding 12 0 6 0 protein RBBP7 IPI00215734 3 PRMT1 Isoform 2 of 12 0 6 0 Protein arginine N- methyltransferase 1 IPI00554648 51 KRT8 Keratin, type II 11 3 6 0 cytoskeletal 8 IPI00027201 64 MUC2 Mucin-2 11 4 6 1 precursor IPI00789401 22 PLS1 PLS1 protein 11 2 6 0 IPI00215743 48 RRBP1 Isoform 3 of 11 3 6 0 Ribosome-binding protein 1 IPI00003881 11 HNRNPF 11 2 6 0 Heterogeneous nuclear ribonucleoprotein F IPI00410017 18 PABPC1 Isoform 2 of 11 2 6 0 Polyadenylate-binding protein 1 IPI00022228 21 HDLBP Vigilin 11 2 6 0 IPI00401264 11 TXNDC4 Thioredoxin 11 2 6 0 domain-containing protein 4 precursor IPI00008438 9 RPS10 40S ribosomal 11 2 6 0 protein S10 IPI00295741 9 CTSB Cathepsin B 11 5 6 0 precursor IPI00004968 7 PRPF19 Pre-mRNA- 11 0 6 1 processing factor 19 IPI00014230 6 C1QBP Complement 11 2 6 0 component 1 Q subcomponent-binding protein, mitochondrial precursor IPI00646917 4 NUDT21 Cleavage and 11 0 6 0 polyadenylation specificity factor subunit 5 IPI00219155 4 RPL27 60S ribosomal 11 1 6 0 protein L27 IPI00218606 4 RPS23 40S ribosomal 11 5 6 0 protein S23 IPI00101405 4 FDPS Farnesyl 11 2 6 0 diphosphate synthase IPI00028006 3 PSMB2 Proteasome 11 2 6 0 subunit beta type-2 IPI00386662 2 FUSIP1 Isoform 4 of 11 1 6 0 FUS-interacting serine- arginine-rich protein 1 IPI00027230 43 HSP90B1 Endoplasmin 10 2 6 0 precursor IPI00027834 15 HNRNPL 10 1 6 0 heterogeneous nuclear ribonucleoprotein L isoform a IPI00219870 23 CTNND1 Isoform 1A of 10 3 6 0 Catenin delta-1 IPI00383296 21 HNRNPM Isoform 2 of 10 2 6 0 Heterogeneous nuclear ribonucleoprotein M IPI00020599 19 CALR Calreticulin 10 2 6 0 precursor IPI00644989 18 PDIA6 Isoform 1 of 10 2 6 0 Protein disulfide- isomerase A6 precursor IPI00646486 9 HP1BP3 10 2 6 0 Heterochromatin protein 1, binding protein 3 IPI00010896 15 CLIC1 Chloride 10 2 6 0 intracellular channel protein 1 IPI00000690 13 AIFM1 Isoform 1 of 10 4 6 0 Apoptosis-inducing factor 1, mitochondrial precursor nPeptides, number of peptides that were identified in all the samples analyzed; nBigT, number of patients in which the ratio (expression level in tumor tissue/expression level in healthy tissue) was >3; nSmallT, number of patients in which the ratio (expression level in tumor tissue/expression level in healthy tissue) was <1/3; nBigP, number of patients in which the ratio (expression level in polyp tissue/expression level in healthy tissue) was >3; nSmallP, number of patients in which the ratio (expression level in polyp tissue/expression level in healthy tissue) was <1/3.

Example 3 Identification of a Group of Protein Markers that are More Highly Expressed in Polyps than in Advanced Stages of Colorectal Cancer

Table 4 lists proteins that were observed to be highly expressed in polyps, whereas in more advanced stages of colorectal cancer these proteins tended to have decreased levels of expression. Accordingly, a diagnostic array of reagents directed to detection of at least some of the proteins in this group may be used as a screening test for very early detection of colorectal cancer. Such a screening test could identify susceptible at-risk individuals, even prior to the stage at which polyp visualization is possible by endoscopic techniques.

TABLE 4 Protein (IPI Acc. No.) nBigTumor nSmallTumor nBigPolyp nSmallPolyp CPT2 Carnitine O- 6 5 12 0 palmitoyltransferase 2, mitochondrial precursor (IPI00012912) ARL1 ADP-ribosylation factor- 6 1 12 0 like protein 1 (IPI00219518) PFKL Isoform 1 of 6- 6 2 11 0 phosphofructokinase, liver type (IPI00332371) GOT2 Aspartate 6 3 11 0 aminotransferase, mitochondrial precursor (IPI00018206) AP1G1 AP-1 complex subunit 6 1 11 0 gamma-1 (IPI00643591) STRBP Isoform 2 of Spermatid 6 0 11 0 perinuclear RNA-binding protein (IPI00413860) CLCA1 Calcium-activated 4 13 11 1 chloride channel regulator 1 precursor (IPI00014625) CYFIP1 Isoform 1 of 4 2 11 0 Cytoplasmic FMR1-interacting protein 1 (IPI00644231) COQ9 Isoform 1 of Ubiquinone 4 3 11 0 biosynthesis protein COQ9, mitochondrial precursor (IPI00470631) NDUFA9 NADH dehydrogenase 3 4 11 0 [ubiquinone] 1 alpha subcomplex subunit 9, mitochondrial precursor (IPI00003968) ALDH7A1 Similar to Antiquitin 2 5 11 0 (IPI00221234) HMGCS1 6 4 10 0 Hydroxymethylglutaryl-CoA synthase, cytoplasmic (IPI00008475) NNT NAD(P) transhydrogenase, 6 2 10 0 mitochondrial precursor (IPI00337541) PRDX5 Uncharacterized protein 6 2 10 0 PRDX5 (Fragment) (IPI00876999) PCCB Propionyl-CoA 6 5 10 0 carboxylase beta chain, mitochondrial precursor (IPI00007247) COPZ1 Coatomer subunit zeta-1 6 4 10 0 (IPI00032851) BAX BCL2-associated X protein 6 2 10 0 isoform sigma (IPI00845474) ACAD9 Acyl-CoA 6 0 10 0 dehydrogenase family member 9, mitochondrial precursor (IPI00152981) UBXD8 UBX domain-containing 6 1 10 0 protein 8 (IPI00172656) HMGCS2 5 12 10 0 Hydroxymethylglutaryl-CoA synthase, mitochondrial precursor (IPI00008934) SLC25A3 cDNA FLJ90278 fis, 5 1 10 0 clone NT2RP1000325, highly similar to Phosphate carrier protein, mitochondrialprecursor (IPI00790115) SLC25A11 Mitochondrial 2- 5 4 10 0 oxoglutarate/malate carrier protein (IPI00219729) PDCD6 Programmed cell death 5 0 10 0 protein 6 (IPI00025277) UCRC Cytochrome b-c1 complex 4 0 10 0 subunit 9 (IPI00554701) DEFA6 Defensin-6 precursor 4 0 10 0 (IPI00008301) DYNC1H1 Cytoplasmic dynein 1 3 1 10 0 heavy chain 1 (IPI00456969) HK1 Isoform 2 of Hexokinase-1 3 1 10 0 (IPI00220663) CYFIP2 Isoform 2 of 3 2 10 0 Cytoplasmic FMR1-interacting protein 2 (IPI00719600) DCI Isoform 2 of 3,2-trans-enoyl- 3 5 10 0 CoA isomerase, mitochondrial precursor (IPI00398758) CISD1 CDGSH iron sulfur 1 1 10 0 domain-containing protein 1 (IPI00020510) nBigTumor, number of patients in which the ratio (expression level in tumor tissue/expression level in healthy tissue) was >3; nSmallTumor, number of patients in which the ratio (expression level in tumor tissue/expression level in healthy tissue) was <1/3; nBigPolyp, number of patients in which the ratio (expression level in polyp tissue/expression level in healthy tissue) was >3; nSmallPolyp, number of patients in which the ratio (expression level in polyp tissue/expression level in healthy tissue) was <1/3.

Example 4 Cancer Associated Proteins Identified by Isotopic Labeling

The proteins identified by isotopic labeling included those listed in Table 5. All of the listed proteins were found to be present in colon cancer tissue at levels that were at least 2.5-fold greater than the level of the same protein in healthy colon tissue from the same subject, as indicated by the median ratios of the protein levels (i.e. cancerous tissue:healthy tissue) listed in the column denoted “MED”.

TABLE 5 Protein Name IPI Acc. No. nPEP MED #>2.5 DPEP1 Dipeptidase 1 precursor IPI00059476 12 50 8 LCN2 Lipocalin 2 IPI00643623 6 48.75 13 FAM62B Isoform 2 of Extended IPI00409635 10 39.73 10 synaptotagmin-2 MTA2 Metastasis-associated IPI00171798 8 31.45 8 protein MTA2 S100A8 Protein S100-A8 IPI00007047 7 30.72 17 S100A9 Protein S100-A9 IPI00027462 7 28.205 17 MPO Isoform H7 of IPI00236556 26 25.57 14 Myeloperoxidase precursor MCM2 DNA replication licensing IPI00184330 12 23.37 11 factor MCM2 FDFT1 Squalene synthetase IPI00020944 5 23.155 7 DMBT1 Isoform 1 of Deleted in IPI00099110 7 22.86 7 malignant brain tumors 1 protein precursor LTF Lactotransferrin precursor IPI00848342 30 20.45 17 SERPINB5 Serpin B5 precursor IPI00783625 7 20.44 8 OLFM4 Olfactomedin-4 precursor IPI00022255 20 19.49 13 FERMT1 Isoform 1 of Fermitin IPI00304754 9 16.05 14 family homolog 1 C1R; ACYP1; C17orf13 IPI00296165 5 15.61 7 Complement C1r subcomponent precursor PLOD3 Procollagen-lysine, 2- IPI00030255 10 15.17 8 oxoglutarate 5-dioxygenase 3 precursor CEACAM5 Carcinoembryonic IPI00027486 9 14.12 17 antigen-related cell adhesion molecule 5 precursor PYCR1 pyrroline-5-carboxylate IPI00376503 6 12.505 12 reductase 1 isoform 2 MCM6 DNA replication licensing IPI00031517 9 12.29 6 factor MCM6 SLC1A5 Neutral amino acid IPI00019472 8 10.03 10 transporter B THBS1 Thrombospondin-1 IPI00296099 13 8.55 14 precursor LACTB2 Beta-lactamase-like IPI00006952 5 8.445 8 protein 2 NAT10 N-acetyltransferase 10 IPI00300127 11 7.85 10 RSL1D1 RSL1D1 protein IPI00642046 11 7.845 12 LMO7 Isoform 3 of LIM domain IPI00291802 11 7.74 10 only protein 7 LYZ Lysozyme C precursor IPI00019038 7 7.64 15 MCM7 Isoform 1 of DNA IPI00299904 10 7.435 7 replication licensing factor MCM7 F11R Junctional adhesion molecule IPI00001754 5 6.775 7 A precursor MCM3 DNA replication licensing IPI00013214 11 6.605 10 factor MCM3 ATP6V1E1 vacuolar H+ ATPase IPI00719806 5 6.34 9 E1 isoform b TNC Isoform 1 of Tenascin IPI00031008 58 6.2 12 precursor COL12A1 Isoform 1 of Collagen IPI00329573 49 6.175 17 alpha-1(XII) chain precursor SORD 11 kDa protein IPI00791243 5 5.78 7 PYCR2 Pyrroline-5-carboxylate IPI00470610 5 5.645 9 reductase 2 GTF2I Isoform 2 of General IPI00293242 10 5.515 7 transcription factor II-I DDX18 ATP-dependent RNA IPI00301323 8 5.38 8 helicase DDX18 RBM39 Isoform 2 of RNA-binding IPI00215801 11 5.19 12 protein 39 NQO1 NAD IPI00012069 6 4.9 8 DNAJA3 Isoform 2 of DnaJ IPI00179187 7 4.73 9 homolog subfamily A member 3, mitochondrial precursor NCBP1 Nuclear cap-binding IPI00019380 8 4.42 9 protein subunit 1 HSPH1 Isoform Beta of Heat IPI00218993 33 4.365 13 shock protein 105 kDa ADSS Adenylosuccinate IPI00026833 7 4.36 11 synthetase isozyme 2 PSAT1 Isoform 1 of Phosphoserine IPI00001734 9 4.295 8 aminotransferase ALG5 Dolichyl-phosphate beta- IPI00002506 6 4.135 10 glucosyltransferase PCNA Proliferating cell nuclear IPI00021700 12 4.04 10 antigen TCOF1 Isoform 2 of Treacle IPI00298696 12 3.92 9 protein SERPINH1 Serpin H1 precursor IPI00032140 20 3.9 16 ERO1L ERO1-like protein alpha IPI00386755 8 3.81 12 precursor ILVBL Isoform 1 of Acetolactate IPI00554541 15 3.8 11 synthase-like protein ANXA3 Annexin A3 IPI00024095 17 3.645 16 NAMPT Isoform 1 of IPI00018873 25 3.61 12 Nicotinamide phosphoribosyltransferase TFRC Transferrin receptor IPI00022462 23 3.56 9 protein 1 SERPINB9 Serpin B9 IPI00032139 8 3.41 10 EIF2S2 Eukaryotic translation IPI00021728 8 3.38 8 initiation factor 2 subunit 2 SRRM2 Isoform 1 of Serine/ IPI00782992 8 3.365 8 arginine repetitive matrix protein 2 ARHGEF1 Isoform 1 of Rho IPI00647786 5 3.31 7 guanine nucleotide exchange factor 1 COMT Isoform Soluble of IPI00375513 9 3.31 10 Catechol O-methyltransferase DEK 48 kDa protein IPI00871695 8 3.285 10 SYK Isoform Long of Tyrosine- IPI00018597 6 3.275 7 protein kinase SYK S100A11 Protein S100-A11 IPI00013895 8 3.2 12 HSDL2 Isoform 1 of IPI00414384 10 3.14 10 Hydroxysteroid dehydrogenase-like protein 2 C7orf24 Uncharacterized protein IPI00031564 5 3.125 9 C7orf24 HM13 Isoform 1 of Minor IPI00152441 7 3.09 9 histocompatibility antigen H13 RCN1 Reticulocalbin-1 precursor IPI00015842 14 3.07 11 DIAPH1 Diaphanous homolog 1 IPI00884341 10 3.06 8 SRM Spermidine synthase IPI00292020 5 2.97 11 ATAD3A Isoform 2 of ATPase IPI00295992 20 2.965 10 family AAA domain-containing protein 3A GPX2 Glutathione peroxidase 2 IPI00298176 9 2.945 9 LOC442497; SLC3A2 solute IPI00554722 13 2.9 12 carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 isoform e SAE1 SUMO-activating enzyme IPI00033130 8 2.89 9 subunit 1 IPO7 Importin-7 IPI00007402 11 2.85 9 SET Isoform 2 of Protein SET IPI00301311 9 2.85 11 PAICS Multifunctional protein IPI00217223 15 2.84 11 ADE2 OCIAD2 Isoform 1 of OCIA IPI00555902 6 2.83 10 domain-containing protein 2 GLT25D1 Glycosyltransferase 25 IPI00168262 5 2.82 7 family member 1 precursor RCC2 Protein RCC2 IPI00465044 12 2.79 10 CTSG Cathepsin G precursor IPI00028064 10 2.77 12 CHD4 Isoform 2 of IPI00455210 10 2.765 8 Chromodomain-helicase- DNA-binding protein 4 SSBP1 Single-stranded DNA- IPI00029744 6 2.765 10 binding protein, mitochondrial precursor ACOT7 Isoform 1 of Cytosolic IPI00010415 6 2.76 8 acyl coenzyme A thioester hydrolase AK3 GTP:AMP IPI00465256 13 2.72 9 phosphotransferase mitochondrial GCA Grancalcin IPI00004524 6 2.71 9 ACIN1 Isoform 1 of Apoptotic IPI00007334 5 2.69 9 chromatin condensation inducer in the nucleus TM9SF4 Isoform 2 of IPI00885106 8 2.69 8 Transmembrane 9 superfamily member 4 precursor CAD Putative uncharacterized IPI00893035 17 2.675 9 protein CAD FASN Fatty acid synthase IPI00026781 74 2.66 11 TJP2 Isoform A1 of Tight junction IPI00003843 16 2.66 8 protein ZO-2 GLRX3 Glutaredoxin-3 IPI00008552 6 2.595 7 RCC1 regulator of chromosome IPI00787306 5 2.59 7 condensation 1 isoform b TOP1 DNA topoisomerase 1 IPI00413611 10 2.57 9 GGH Gamma-glutamyl hydrolase IPI00023728 11 2.565 10 precursor PUF60 Isoform 5 of Poly IPI00856076 10 2.56 9 TPR nuclear pore complex- IPI00742682 16 2.545 9 associated protein TPR GMPS GMP synthase IPI00029079 11 2.525 10 PKP3 Plakophilin-3 IPI00026952 19 2.52 10 LOC731605 similar to BCL2- IPI00886854 7 2.515 9 associated transcription factor 1 isoform 2 SLC2A1 Solute carrier family 2, IPI00220194 8 2.515 7 facilitated glucose transporter member 1 C8orf55 Uncharacterized protein IPI00171421 7 2.51 8 C8orf55 precursor HCFC1 Uncharacterized protein IPI00641743 7 2.51 10 HCFC1 IPI Acc. No, accession number in IPI database; nPEP, number of identified peptides in all the samples, MED, median of the ratio tumor/healthy from all the patients; #>2.5, number of patients in which tumor/healthy >2.5.

Example 5 Cancer Associated Proteins Identified by Multidimensional Chromatography

The proteins identified on the basis of the peptides detected by the multidimensional chromatography technique include those listed in Table 6, and reflects the abundance of these proteins in cancerous tissues. None of the proteins listed in Table 6 were identified by the isotopic labeling technique. The proteins listed in Tables 3 and 6 appear to be specifically expressed in colorectal cancer tissue, as they were substantially undetectable in all healthy colorectal tissues obtained from the colorectal cancer patients.

TABLE 6 IPI Acc. No. of Protein Name No. AVG SD tumors ADAMDEC1 ADAM DEC1 precursor 4480 50 0 AMACR Alpha-methylacyl-CoA 847727 50 0 7 racemase AMACR; C1QTNF3 alpha-methylacyl- 5918 50 0 7 CoA racemase isoform 1 ARID1A Isoform 1 of AT-rich interactive 643722 50 0 domain-containing protein 1A CEBPZ CCAAT/enhancer-binding 306723 50 0 protein zeta COL5A1 Collagen alpha-1(V) chain 844090 50 0 precursor EFEMP2 Mutant p53 binding protein 1 556657 50 0 variant (Fragment) FAM84B Protein FAM84B 64666 50 0 FKBP10 FK506-binding protein 10 303300 50 0 8 precursor FKBP9 FK506-binding protein 9 182126 50 0 9 precursor GPRC5A Retinoic acid-induced protein 3 22624 50 0 8 KPNA2 Karyopherin alpha 2 789457 50 0 MMP1 Interstitial collagenase precursor 8561 50 0 PNMA5 Paraneoplastic antigen-like 514588 50 0 protein 5 POLR1C Isoform 1 of DNA-directed 5179 50 0 9 RNA polymerases I and III subunit RPAC1 SPARC SPARC precursor 14572 50 0 5 UBAP2 Ubiquitin-associated protein 2 171127 50 0 5 UCK2 Isoform 1 of Uridine-cytidine 65671 50 0 kinase 2 WDR74 Isoform 1 of WD repeat- 18192 50 0 containing protein 74

Example 6 Cancer Associated Proteins as Potential Therapeutic Targets

Table 7 lists proteins that are considered potential targets for development of cytotoxic reagents specifically directed to these proteins, for example, specific antibody or antibody fragments conjugated to toxic moieties for targeted elimination of cancer cells. As well as being highly expressed in early stage polyps and in tumors, these proteins are generally exposed, and considered vulnerable to attack by targeted cytotoxic reagents.

TABLE 7 Protein (IPI Acc. No.) nBigTumor nSmallTumor nBigPolyp nSmallPolyp Ontology DEFA3 Neutrophil 26 2 5 0 EX defensin 3 precursor (IPI00021827) ELA2 Leukocyte 23 0 6 0 EX elastase precursor (IPI00027769) LYZ Lysozyme C 17 2 8 0 EX precursor (IPI00019038) LOC442497; SLC3A2 16 1 8 0 PM solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 isoform e (IPI00554722) SLC1A5 Neutral 16 0 6 0 PM amino acid transporter B (IPI00019472) DMBT1 Isoform 1 of 15 0 8 1 EX AM Deleted in malignant brain tumors 1 protein precursor (IPI00099110) NUCB1 Nucleobindin- 15 3 7 0 EX AM 1 precursor (IPI00295542) SLC12A2 Isoform 1 of 14 2 16 0 PM Solute carrier family 12 member 2 (IPI00022649) GGH Gamma- 14 1 12 0 EX glutamyl hydrolase precursor (IPI00023728) AGR3 Anterior 14 4 7 0 EX gradient protein 3 homolog precursor (IPI00152409) MARCKSL1 13 0 11 0 PM MARCKS-related protein (IPI00641181) TM9SF2 12 1 9 0 PM Transmembrane 9 superfamily member 2 precursor (IPI00018415) SYK Isoform Long of 11 0 10 0 PM Tyrosine-protein kinase SYK (IPI00018597) GCA Grancalcin 11 0 7 0 PM (IPI00004524) HDLBP Vigilin 11 2 6 0 PM (IPI00022228) C1QBP Complement 11 2 6 0 PM component 1 Q subcomponent-binding protein, mitochondrial precursor (IPI00014230) KIAA0152 10 0 13 0 PM Uncharacterized protein KIAA0152 precursor (IPI00029046) CLIC1 Chloride 10 2 6 0 PM intracellular channel protein 1 (IPI00010896) nBigTumor, number of patients in which the ratio (expression level in tumor tissue/expression level in healthy tissue) was >3; nSmallTumor, number of patients in which the ratio (expression level in tumor tissue/expression level in healthy tissue) was <1/3; nBigPolyp, number of patients in which the ratio (expression level in polyp tissue/expression level in healthy tissue) was >3; nSmallPolyp, number of patients in which the ratio (expression level in polyp tissue/expression level in healthy tissue) was <1/3; EX, extracellular region; PM, plasma membrane; AM, additional membrane.

REFERENCES CITED

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The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the brand concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention. 

1-46. (canceled)
 47. A method for detecting a colorectal pre-cancerous tissue in a subject, comprising the steps of: (i) contacting a colorectal tissue with a pharmaceutical composition comprising a reagent suitable for detecting a combination of markers; and (ii) detecting in said colorectal tissue the presence of said combination, wherein said combination comprises: (a) KIAA0152, NAMPT, or both; and (b) PYCR1, GPX2, PRKDC, ALDH18A1, OCIAD2, GCS1, GMDS, ARF4, ARF5, LRPPRC, CTNNB1, ARF3, GCN1L1, BDH1, RPL9, UGCGL1, FAM3D, CCT4, CPT2, ARL1, PFKL, GOT2, AP1G1, STRBP, CLCA1, CYFIP1, COQ9, NDUFA9, ALDH7A1, HMGCS1, NNT, PRDX5, PCCB, COPZ1, BAX, ACAD9, UBXD8, HMGCS2, SLC25A, PDCD6, UCRC, DEFA6, DYNC1H1, HK1, CYFIP2, DCI, S100A8, S100A9, CEACAM5, LTF, COL12A1, MPO, DEFA3, GGH, CISD1, ELA2, SLC12A2, VDAC1, AGR2, PIGR, CTSG, CTNNB1, AGR3, TNC, APOB48R, CDH17, FCGBP, CDH1, GPA33, ANXA3, OLFM4, LCN2, SERPINB5, DSG2, COPA, HSD17B11, ATP2A2, CDH1, or any combination thereof, thereby detecting a colorectal pre-cancerous tissue in a subject.
 48. The method according to claim 47, wherein said detecting in a colorectal tissue the presence is detecting in a colorectal tissue an expression level above the expression level of a reference colorectal tissue.
 49. The method according to claim 47, wherein said reference colorectal tissue is a non-diseased tissue.
 50. The method according to claim 47, further comprising the step of obtaining a colorectal tissue sample prior to said detecting.
 51. The method according to claim 50, wherein said sample is obtained from said subject by a procedure selected from the group consisting of biopsy, flexible endoscopy, double balloon endoscopy and surgical colorectal re-sectioning.
 52. The method according to claim 47, wherein said detecting is detecting in-vivo.
 53. The method according to claim 47, wherein said detecting is detecting ex vivo.
 54. The method according to claim 52, wherein said detecting in-vivo is detecting in an intact colorectal tissue.
 55. The method according to claim 47, wherein said reagent comprises an antibody, or an antibody mimetic.
 56. The method according to claim 47, wherein said reagent comprises a bi-specific antibody.
 57. The method according to claim 47, wherein said pharmaceutical composition comprises a chemotherapeutic agent.
 58. The method according to claim 47, wherein said detecting is performing an assay system selected from the group consisting of an immunoassay, a nucleic acid hybridization assay, a binding assay, an array, a phage display library or any combination thereof.
 59. The method according to claim 48, wherein said above is at least 3-fold above.
 60. The method according to claim 47, wherein said subject is at risk for developing colorectal cancer.
 61. The method according to claim 47, wherein said pre-cancerous tissue is a pre-cancerous polyp.
 62. The method according to claim 47, wherein said reference colorectal tissue is from said subject.
 63. The method according to claim 47, wherein said detecting is detecting a fluorescent dye.
 64. A method for determining the stage of a cancerous or pre-cancerous growth in a subject, the method comprising: (i) detecting in a test sample from the subject a plurality of proteins selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20), so as to determine the level of the at least one protein; and (ii) comparing the level determined in (i) to a reference level of the same at least one protein; wherein the level detected in the test sample is indicative of the stage of the growth.
 65. A method for determining the prognosis of a cancerous disease in a subject, the method comprising: (i) detecting in a test sample from the subject a plurality of proteins selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20), so as to determine the level of the at least one protein; and (ii) comparing the level determined in (i) to a reference level of the same at least one protein; wherein the level detected in the test sample is indicative of the prognosis of the cancerous disease. 